Sequences of hepatitis C virus genotypes and their use as therapeutic and diagnostic agents

ABSTRACT

The present invention relates to a polynucleic acid composition comprising or consisting of at least one polynucleic acid containing 8 or more contiguous nucleotides corresponding to a nucleotide sequence from the region spanning positions 417 to 957 of the Core/E1 region of HCV type 3; and/or the region spanning positions 4664 to 4730 of the NS3 region of HCV type 3; and/or the region spanning positions 4892 to 5292 of the NS3/4 region of HCV type 3; and/or the region spanning positions 8 023 to 8 235 of the NS5 region of the BR36 subgroup of HCV type 3 a ; and/or the coding region of HCV type 4 a  starting at nucleotide 379 in the core region; and/or the coding region of HCV type 4; and/or the coding region of HCV type 5, with said nucleotide numbering being with respect to the numbering of HCV nucleic acids as shown in Table 1, and with said polynucleic acids containing at least one nucleotide difference with known HCV type 1, and/or HCV type 2 genomes in the above-indicated regions, or the complement thereof.

This application is a divisional of application Ser. No. 08/362,455,filed Jan. 11, 1995, which is a 371 U.S. national phase ofPCT/EP94/01323, filed Apr. 27, 1994, which designated the U.S., theentire content of which is hereby incorporated by reference in thisapplication.

The invention relates to new sequences of hepatitis C virus (HCV)genotypes and their use as therapeutic and diagnostic agents.

The present invention relates to new nucleotide and amino acid sequencescorresponding to the coding region of a new type 2 subtype 2d,type-specific sequences corresponding to HCV type 3a, to new sequencescorresponding to the coding region of a new subtype 3c, and to newsequences corresponding to the coding region of HCV type 4 and type 5subtype 5a; a process for preparing them, and their use for diagnosis,prophylaxis and therapy.

The technical problem underlying the present invention is to provide newtype-specific sequences of the Core, the E1, the E2, the NS3, the NS4and the NS5 regions of HCV type 4 and type 5, as well as of new variantsof HCV types 2 and 3. These new HCV sequences are useful to diagnose thepresence of type 2 and/or type 3 and/or type 4 and/or type 5 HCVgenotypes in a biological sample. Moreover, the availability of thesenew type-specific sequences can increase the overall sensitivity of HCVdetection and should also prove to be useful for therapeutic purposes.

Hepatitis C viruses (HCV) have been found to be the major cause ofnon-A, non-B hepatitis. The sequences of cDNA clones covering thecomplete genome of several prototype isolates have been determined (Katoet al., 1990; Choo et al., 1991; Okamoto et al., 1991; Okamoto et al.,1992). Comparison of these isolates shows that the variability innucleotide sequences can be used to distinguish at least 2 differentgenotypes, type 1 (HCV-1 and HCV-J) and type 2 (HC-J6 and HC-J8), withan average homology of about 68%. Within each type, at least twosubtypes exist (e.g. represented by HCV-1 and HCV-J), having an averagehomology of about 79%. HCV genomes belonging to the same subtype showaverage homologies of more than 90% (Okamoto et al., 1992). However, thepartial nucleotide sequence of the NS5 region of the HCV-T isolatesshowed at most 67% homology with the previously published sequences,indicating the existence of a yet another HCV type (Mori et al., 1992).Parts of the 5′ untranslated region (UR), core, NS3, and NS5 regions ofthis type 3 have been published, further establishing the similarevolutionary distances between the 3 major genotypes and their subtypes(Chan et al., 1992).

The identification of type 3 genotypes in clinical samples can beachieved by means of PCR with type-specific primers for the NS5 region.However, the degree to which this will be successful is largelydependent on sequence variability and on the virus titer present in theserum. Therefore, routine PCR in the open reading frame, especially fortype 3 and the new type 4 and 5 described in the present inventionand/or group V (Cha et al., 1992) genotypes can be predicted to beunsuccessful. A new typing system (LiPA), based on variation in thehighly conserved 5′ UR, proved to be more useful because the 5 major HCVgenotypes and their subtypes can be determined (Stuyver et al., 1993).The selection of high-titer isolates enables to obtain PCR fragments forcloning with only 2 primers, while nested PCR requires that 4 primersmatch the unknown sequences of the new type 3, 4 and 5 genotypes.

New sequences of the 5′ untranslated region (5′UR) have been listed byBukh et al. (1992). For some of these, the E1 region has recently beendescribed (Bukh et al., 1993). Isolates with similar sequences in the5′UR to a group of isolates including DK12 and HK10 described by Bukh etal. (1992) and E-b1 to E-b8 described and classified as type 3 by Chanet al. (1991), have been reported and described in the 5′UR, thecarboxyterminal part of E1, and in the NS5 region as group IV by Cha etal. (1992; WO 92/19743), and have also been described in the 5′UR forisolate BR56 and classified as type 3 by the inventors of thisapplication (Stuyver et al., 1993).

The aim of the present invention is to provide new HCV nucleotide andamino acid sequences enabling the detection of HCV infection.

Another aim of the present infection is to provide new nucleotide andamino acid HCV sequences enabling the classification of infectedbiological fluids into different serological groups unambiguously linkedto types and subtypes at the genome level.

Another aim of the present invention is to provide new nucleotide andamino acid HCV sequences ameliorating the overall HCV detection rate.

Another aim of the present invention is to provide new HCV sequences,useful for the design of HCV vaccine compositions.

Another aim of the present invention is to provide a pharmaceuticalcomposition consisting of antibodies raised against the polypeptidesencoded by these new HCV sequences, for therapy or diagnosis.

The present invention relates more particularly to a compositioncomprising or consisting of at least one polynucleic acid containing atleast 5, and preferably 8 or more contiguous nucleotides selected fromat least one of the following HCV sequences:

-   -   an HCV type 3 genomic sequence, more particularly in any of the        following regions:        -   the region spanning positions 417 to 957 of the Core/E1            region of HCV subtype 3a,        -   the region spanning positions 4664 to 4730 of the NS3 region            of HCV type 3,        -   the region spanning positions 4892 to 5292 of the NS3/4            region of HCV type 3,        -   the region spanning positions 8023 to 8235 of the NS5 region            of the BR36 subgroup of HCV subtype 3a,        -   an HCV subtype 3c genomic sequence, more particularly the            coding regions of the above-specified regions;    -   an HCV subtype 2d genomic sequence, more particularly the coding        region of HCV subtype 2d,    -   an HCV type 4 genomic sequence, more particularly the coding        region, more particularly the coding region of subtypes 4a, 4e,        4f, 4g, 4h, 4i, and 4j,    -   an HCV type 5 genomic sequence, more particularly the coding        region of HCV type 5, more particularly the regions encoding        Core, E1, E2, NS3, and NS4

with said nucleotide numbering being with respect to the numbering ofHCV nucleic acids as shown in Table 1, and with said polynucleic acidscontaining at least one nucleotide difference with known HCV (type 1,type 2, and type 3) polynucleic acid sequences in the above-indicatedregions, or the complement thereof.

It is to be noted that the nucleotide difference in the polynucleicacids of the invention may involve or not an amino acid difference inthe corresponding amino acid sequences coded by said polynucleic acids.

According to a preferred embodiment, the present invention relates to acomposition comprising or containing at least one polynucleic acidencoding an HCV polyprotein, with said polynucleic acid containing atleast 5, preferably at least 8 nucleotides corresponding to at leastpart of an HCV nucleotide sequence encoding an HCV polyprotein, and withsaid HCV polyprotein containing in its sequence at least one of thefollowing amino acid residues: L7, Q43, M44, S60, R67, Q70, T71, A79,A87, N106, K115, A127, A190, S130, V134, G142, I144, E152, A157, V158,P165, S177 or Y177, I178, V180 or E180 or F182, R184, I186, H187, T189,A190, S191 or G191, Q192 or L192 or I192 or V192 or E192, N193 or H193or P193, W194 or Y194, H195, A197 or I197 or V197 or T197, V202, I203 orL203, Q208, A210, V212, F214, T216, R217 or D217 or E217 or V217, H218or N218, H219 or V219 or L219, L227 or I227, M231 or E231 or Q231, T232or D232 or A232 or K232, Q235 or I235, A237 or T237, I242, I246, S247,S248, V249, S250 or Y250, I251 or V251 or M251 or F251, D252, T254 orV254, L255 or V255, E256 or A256, M258 or F258 or V258, A260 or Q260 orS260, A261, T264 or Y264, M265, I266 or A266, A267, G268 or T268, F271or M271 or V271, I277, M280 or H280, I284 or A284 or L84, V274, V291,N292 or S292, R293 or I293 or Y293, Q294 or R294, L297 or I297 or Q297,A299 or K299 or Q299, N303 or T303, T308 or L308, T310 or F310 or A310or D310 or V310, L313, G317 or Q317, L333, S351, A358, A359, A363, S364,A366, T369, L373, F376, Q386, I387, S392, I399, F402, I403, R405, D454,A461, A463, T464, K484, Q500, E501, S521, K522, H524, N528, S531, S532,V534, F536, F537, M539, I546, C1282, A1283, H1310, V1312, Q1321, P1368,V1372, V1373, K1405, Q1406, S1409, A1424, A1429, C1435, S1436, S1456,H1496, A1504, D1510, D1529, I1543, N1567, D1556, N1567, M1572, Q1579,L1581, S1583, F1585, V1595, E1606 or T1606, M1611, V1612 or L1612,P1630, C1636, P1651, T1656 or I1656, L1663, V1667, V1677, A1681, H1685,E1687, G1689, V1695, A1700, Q1704, Y1705, A1713, A1714 or S1714, M1718,D1719, A1721 or T1721, R1722, A1723 or V1723, H1726 or G1726, E1730,V1732, F1735, I1736, S1737, R1738, T1739, G1740, Q1741, K1742, Q1743,A1744, T1745, L1746, E1747 or K1747, I1749, A1750, T1751 or A1751,V1753, N1755, K1756, A1757, P1758, A1759, H1762, T1763, Y1764, P2645,A2647, K2650, K2653 or L2653, S2664, N2673, F2680, K2681, L2686, H2692,Q2695 or L2695 or I2695, V2712, F2715, V2719 or Q2719, T2722, T2724,S2725, R2726, G2729, Y2735, H2739, I2748, G2746 or I2746, I2748, P2752or K2752, P2754 or T2754, T2757 or P2757, with said notation beingcomposed of a letter representing the amino acid residue by itsone-letter code, and a number representing the amino acid numberingaccording to Kato et al., 1990.

Each of the above-mentioned residues can be found in any of FIGS. 2, 5,7, 11 or 12 showing the new amino acid sequences of the presentinvention aligned with known sequences of other types or subtypes of HCVfor the Core, E1, E2, NS3, NS4, and NS5 regions.

More particularly, a polynucleic acid contained in the compositionaccording to the present invention contains at least 5, preferably 8, ormore contiguous nucleotides corresponding to a sequence of contiguousnucleotides selected from at least one of HCV sequences encoding thefollowing new HCV amino acid sequences:

-   -   new sequences spanning amino acid positions 1 to 319 of the        Core/E1 region of HCV subtype 2d, type 3 (more particularly new        sequences for subtypes 3a and 3c), new type 4 subtypes (more        particularly new sequences for subtypes 4a, 4e, 4f, 4g, 4h, 4i        and 4j) and type 5a, as shown in FIG. 5;    -   new sequences spanning amino acid positions 328 to 546 of the        E1/E2 region of HCV subtype 5a as shown in FIG. 12;    -   new sequences spanning amino acid positions 1556 to 1764 of the        NS3/NS4 region of HCV type 3 (more particularly for new subtypes        3a sequences), and subtype 5a, as shown in FIG. 7 or 11;    -   new sequences spanning amino acid positions 2645 to 2757 of the        NS5B region of HCV subtype 2d, type 3 (more particularly for new        subtypes 3a and 3c), new type 4 subtypes (more particularly        subtypes 4a, 4e, 4f, 4g, 4h, 4i and 4j) and subtype 5a, as shown        in FIG. 2,

Using the LiPA system mentioned above, Brazilian blood donors with hightiter type 3 hepatitis C virus, Gabonese patients with high-titer type 4hepatitis C virus, and a Belgian patient with high-titer HCV type 5infection were selected. Nucleotide sequences in the core, E1, NS5 andNS4 regions which have not yet been reported before, were analyzed inthe frame of the invention. Coding sequences (with the exception of thecore region) of any type 4 isolate are reported for the first time inthe present invention. The NS5b region was also analyzed for the newtype 3 isolates. After having determined the NS5b sequences, comparisonwith the Ta and Tb subtypes described by Mori et al. (1992) waspossible, and the type 3 sequences could be identified as type 3agenotypes. The new type 4 isolates segregated into 10 subtypes, based onhomologies obtained in the NS5 and E1 regions. New type 2 and 3sequences could also be distinguished from previously described type 2or 3 subtypes from sera collected in Belgium and the Netherlands.

The term “polynucleic acid” refers to a single stranded or doublestranded nucleic acid sequence which may contain at least 5 contiguousnucleotides to the complete nucleotide sequence (f.i. at least 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or more contiguous nucleotides). A polynucleicacid which is up till about 100 nucleotides in length is often alsoreferred to as an oligonucleotide. A polynucleic acid may consist ofdeoxyribonucleotides or ribonucleotides, nucleotide analogues ormodified nucleotides, or may have been adapted for therapeutic purposes.A polynucleic acid may also comprise a double stranded cDNA clone whichcan be used for cloning purposes, or for in vivo therapy, orprophylaxis.

The term “polynucleic acid composition” refers to any kind ofcomposition comprising essentially said polynucleic acids. Saidcomposition may be of a diagnostic or a therapeutic nature.

The expression “nucleotides corresponding to” refers to nucleotideswhich are homologous or complementary to an indicated nucleotidesequence or region within a specific HCV sequence.

The term “coding region” corresponds to the region of the HCV genomethat encodes thy HCV polyprotein. In fact, it comprises the completegenome with the exception of the 5′ untranslated region and 3′untranslated region.

The term “HCV polyprotein” refers to the HCV polyprotein of the HCV-Jisolate (Kato et al., 1990). The adenine residue at position 330 (Katoet al., 1990) is the first residue of the ATG codon that initiates thelong HCV polyprotein of 3010 amino acids in HCV-J and other type 1bisolates, and of 3011 amino acids in HCV-1 and other type 1a isolates,and of 3033 amino acids in type 2 isolates HC-J6 and HC-J8 (Okamoto etal., 1992).

This adenine is designated as position 1 at the nucleic acid level, andthis methionine is designated as position 1 at the amino acid level, inthe present invention. As type 1a isolates contain 1 extra amino acid inthe NS5a region, coding sequences of type 1a and 1b have identicalnumbering in the Core, E1, NS3, and NS4 region, but will differ in theNS5b region as indicated in Table 1. Type 2 isolates have 4 extra aminoacids in the E2 region, and 17 or 18 extra amino acids in the NS5 regioncompared to type 1 isolates, and will differ in numbering from type 1isolates in the NS3/4 region and NS5b regions as indicated in Table 1.

TABLE 1 Positions Positions Positions Positions described in describedfor described for described for the HCV-J HCV-1 HC-J6, HC-J8 present(Kato et al., (Choo et al., (Okamoto et Region invention* 1990) 1991)al., 1992) Nucleotides NS5b 8023/8235 8352/8564 8026/8238 8433/86457932/8271 8261/8600 7935/8274 8342/8681 NS3/4 4664/5292 4993/56214664/5292 5017/5645 4664/4730 4993/5059 4664/4730 5017/5083 4892/52925221/5621 4892/5292 5245/5645 3856/4209 4185/4528 3856/4209 4209/47624936/5292 5265/5621 4936/5292 5289/5645 coding  330/9359   1/9033 342/9439 region of present invention Amino NS5b 2675/2745 2675/27452676/2746 2698/2768 Acids 2645/2757 2645/2757 2646/2758 2668/2780 NS3/41556/1764 1556/1764 1556/1764 1560/1768 1286/1403 1286/1403 1286/14031290/1407 1646/1764 1646/1764 1646/1764 1650/1768Table 1: Comparison of the HCV nucleotide and amino acid numberingsystem used in the present invention (*) with the numbering used forother prototype isolates. For example, 8352/8564 indicates the regiondesignated by the numbering from nucleotide 8352 to nucleotide 8564 asdescribed by Kato et al. (1990). Since the numbering system of thepresent invention starts at the polyprotein initiation site, the 329nucleotides of the 5′ untranslated region described by Kato et al (1990)have to be substracted, and the corresponding region is numbered fromnucleotide 8023 (“8352-329”) to 8235 (“8564-329”)

The term “HCV type” corresponds to a group of HCV isolates of which thecomplete genome shows more than 74% homology at the nucleic acid level,or of which the NS5 region between nucleotide positions 7932 and 8271shows more than 74% homology at the nucleic acid level, or of which thecomplete HCV polyprotein shows more than 78% homology at the amino acidlevel, or of which the NS5 region between amino acids at positions 2645and 2757 shows more than 80% homology at the amino acid level, topolyproteins of the other isolates of the group, with said numberingbeginning at the first ATG codon or first methionine of the long HCVpolyprotein of the HCV-J isolate (Kato et al., 1990). Isolates belongingto different types of HCV exhibit homologies, over the complete genome,of less than 74% at the nucleic acid level and less than 78% at theamino acid level. Isolates belonging to the same type usually showhomologies of about 92 to 95% at the nucleic acid level and 95 to 96% atthe amino acid level when belonging to the same subtype, and thosebelonging to the same type but different subtypes preferably showhomologies of about 79% at the nucleic acid level and 85–86% at theamino acid level.

More preferably the definition of HCV types is concluded from theclassification of HCV isolates according to their nucleotide distancescalculated as detailed below

(1) based on phylogenetic analysis of nucleic acid sequences in the NS5bregion between nucleotides 7935 and 8274 (Choo et al., 1991) or 8261 and8600 (Kato et al., 1990) or 8342 and 8681 (Okamoto et al., 1991),isolates belonging to the same HCV type show nucleotide distances ofless than 0.34, usually less than 0.33, and more usually of less than0.32, and isolates belonging to the same subtype show nucleotidedistances of less than 0.135, usually of less than 0.13, and moreusually of less than 0.125, and consequently isolates belonging to thesame type but different subtypes show nucleotide distances ranging from0 135 to 0.34, usually ranging from 0.1384 to 0.2477, and more usuallyranging from 0.15 to 0.32, and isolates belonging to different HCV typesshow nucleotide distances greater than 0.34, usually greater that 0.35,and more usually of greater than 0.358, more usually ranging from 0 1384to 0.2977.

(2) based on phylogenetic analysis of nucleic acid sequences in thecore/E1 region between nucleotides 378 and 957, isolates belonging tothe same HCV type show nucleotide distances of less than 0.38, usuallyof less than 0.37, and more usually of less than 0.364, and isolatesbelonging to the same subtype show nucleotide distances of less than0.17, usually of less than 0.16, and more usually of less than 0.15,more usually less than 0.135, more usually less than 0.134, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.15 to 0.38, usually rangingfrom 0.16 to 0.37, and more usually ranging from 0.17 to 0.36, moreusually ranging from 0.133 to 0.379, and isolates belonging to differentHCV types show nucleotide distances greater than 0.34, 0.35, 0.36,usually more than 0.365, and more usually of greater than 0.37,

(3) based on phylogenetic analysis of nucleic acid sequences in theNS3/NS4 region between nucleotides 4664 and 5292 (Choo et al., 1991) orbetween nucleotides 4993 and 5621 (Kato et al., 1990) or betweennucleotides 5017 and 5645 (Okamoto et al., 1991), isolates belonging tothe same HCV type show nucleotide distances of less than 0.35, usuallyof less than 0.34, and more usually of less than 0.33, and isolatesbelonging to the same subtype show nucleotide distances of less than0.19, usually of less than 0.18, and more usually of less than 0.17, andconsequently isolates belonging to the same type but different subtypesshow nucleotide distances ranging from 0.17 to 0.35, usually rangingfrom 0.18 to 0 34, and more usually ranging from 0.19 to 0.33, andisolates belonging to different HCV types show nucleotide distancesgreater than 0.33, usually greater than 0.34, and more usually ofgreater than 0.35.

TABLE 2 Molecular evolutionary distances Core/E1 E1 NS5B NS5B Region 579bp 384 bp 340 bp 222 bp Isolates* 0.0017 − 0.1347 0.0026 − 0.2031 0.0003− 0.1151  0.000 − 0.1323 (0.0750 ± 0.0245) (0.0969 ± 0.0289) (0.0637 ±0.0229) (0.0607 ± 0.0205) Subtypes* 0.1330 − 0.3794 0.1645 − 0.48690.1384 − 0.2977  0.117 − 0.3538 (0.2786 ± 0.0363) (0.3761 ± 0.0433)(0.2219 ± 0.0341) (0.2391 ± 0.0399) Types* 0.3479 − 0.6306 0.4309 −0.9561 0.3581 − 0.6670 0.3457 − 0.7471 (0.4703 ± 0.0525) (0.6308 ±0.0928) (0.4994 ± 0.0495) (0.5295 ± 0.0627) *Figures created by thePHYLIP program DNADIST are expressed as minimum to maximum (average ±standard deviation). Phylogenetic distances for isolates belonging tothe same subtype (‘isolates’), to different subtypes of the same type(‘subtypes’), and to different types (‘types’) are given.

In a comparative phylogenetic analysis of available sequences, ranges ofmolecular evolutionary distances for different regions of the genomewere calculated, based on 19,781 pairwise comparisons by means of theDNA DIST program of the phylogeny inference package PHYLIP version 3.5C(Felsenstein, 1993). The results are shown in Table 2 and indicate thatalthough the majority of distances obtained in each region fit withclassification of a certain isolate, only the ranges obtained in the 340bp NS5B-region are non-overlapping and therefor conclusive. However, aswas performed in the present invention, it is preferable to obtainsequence information from at least 2 regions before final classificationof a given isolate.

Designation of a number to the different types of HCV and HCV typesnomenclature is based on chronological discovery of the different types.The numbering system used in the present invention might still fluctuateaccording to international conventions or guidelines. For example, “type4” might be changed into “type 5” or “type 6”.

The term “subtype” corresponds to a group of HCV isolates of which thecomplete polyprotein shows a homology of more than 90% both at thenucleic acid and amino acid levels, or of which the NS5 region betweennucleotide positions 7932 and 8271 shows a homology of more than 90% atthe nucleic acid level to the corresponding parts of the genomes of theother isolates of the same group, with said numbering beginning with theadenine residue of the initiation codon of the HCV polyprotein. Isolatesbelonging to the same type but different subtypes of HCV show homologiesof more than 74% at the nucleic acid level and of more than 78% at theamino acid level.

The term “BR36 subgroup” refers to a group of type 3a HCV isolates(BR36, BR33, BR34) that are 95%, preferably 95.5%, most preferably 96%homologous to the sequences as represented in SEQ ID NO 1, 3, 5, 7, 9,11 in the NS5b region from position 8023 to 8235.

It is to be understood that extremely variable regions like the E1, E2and NS4 regions will exhibit lower homologies than the average homologyof the complete genome of the polyprotein.

Using these criteria, HCV isolates can be classified into at least 6types. Several subtypes can clearly be distinguished in types 1, 2, 3and 4: 1a, 1b, 2a, 2b, 2c, 2d, 3a, 3b, 4a, 4c, 4d, 4e, 4f, 4g, 4h, 4iand 4j based on homologies of the 5′ UR and coding regions including thepart of NS5 between positions 7932 and 8271. An overview of most of thereported isolates and their proposed classification according to thetyping system of the present invention as well as other proposedclassifications is presented in Table 3.

TABLE 3 HCV CLASSIFICATION OKA- NAKA MOTO MORI O CHA PROTOTYPE 1a I I PtGI HCV-1, HCV-H, HC-J1 1b II II KI GIII HCV-J, HCV-BK, HCV-T, HC-JK1,HC- J4, HCV-CHINA 1c HC-G9 2a III III K2a GIII HC-J6 2b IV IV K2b GIIIHC-J8 2c S83, ARG6, ARG8, I10, T983 2d NE92 3a V V K3 GIV E-b1, Ta,BR36, BR33, HD10, NZL1 3b VI K3 GIV HCV-TR, Tb 3c BE98 4a Z4, GB809-4 4bZ1 4c GB116, GB358, GB215, Z6, Z7 4d DK13 4e GB809-2, CAM600, CAM736 4fCAM622, CAM627 4g GB549 4h GB438 4i CAR4/1205 4j CAR1/501 4k EG29 5a GVSA3, SA4, SA1, SA7, SA11, BE95 6a HK1, HK2, HK3, HK4

The term “complement” refers to a nucleotide sequence which iscomplementary to an indicated sequence and which is able to hybridize tothe indicated sequences.

The composition of the invention can comprise many combinations. By wayof example, the composition of the invention can comprise:

-   -   two (or more) nucleic acids from the same region or,    -   two nucleic acids (or more), respectively from different        regions, for the same isolate or for different isolates,    -   or nucleic acids from the same regions and from at least two        different regions (for the same isolate or for different        isolates).

The present invention relates more particularly to a polynucleic acidcomposition as defined above, wherein said polynucleic acid correspondsto a nucleotide sequence selected from any of the following HCV type 3genomic sequences:

-   -   an HCV genomic sequence having a homology of at least 67%,        preferably more than 69%, more preferably 71%, even more        preferably more than 73%, or most preferably more than 76% to        any of the sequences as represented in SEQ ID NO 13, 15, 17, 19,        21, 23, 25 or 27 (HD10, BR36 or BR33 sequences) in the region        spanning positions 417 to 957 of the Core/E1 region as shown in        FIG. 4;    -   an HCV genomic sequence having a homology of at least 65%,        preferably more than 67%, preferably more than 69%, even        preferably more than 70%, most preferably more than 74% to any        of the sequences as represented in SEQ ID NO 13, 15, 17, 19, 21,        23, 25 or 27 (HD10, BR36 or BR33 sequences) in the region        spanning positions 574 to 957 of the E1 region as shown in FIG.        4;    -   an HCV genomic sequence as having a homology of at least 79%,        more preferably at least 81%, most preferably more than 83% or        more to any of the sequences as represented in SEQ ID NO 147        (representing positions 1 to 346 of the Core region of HVC type        3c, sequence BE98) in the region spanning positions 1 to 378 of        the Core region as shown in FIG. 3;    -   an HCV genomic sequence of HVC type 3a having a homology of at        least 74%, more preferably at least 76%, most preferably more        than 78% or more to any of the sequences as represented in SEQ        ID NO 13, 15, 17, 19, 21, 23, 25 or 27 (HD10, BR36 or BR33        sequences) in the region spanning positions 417 to 957 in the        Core/E1 region as shown in FIG. 4;    -   an HCV genomic sequence of HCV type 3a as having a homology of        at least 74%, preferably more than 76%, most preferably 78% or        more to any of the sequences as represented in SEQ ID NO 13, 15,        17, 19, 21, 23, 25 or 27 (HD10, BR36 or BR33 sequences) in the        region spanning positions 574 to 957 in the E1 region as shown        in FIG. 4;    -   an HCV genomic sequence as having a homology of more than 73.5%,        preferably more than 74%, most preferably 75% homology to the        sequence as represented in SEQ ID NO 29 (HCC153 sequence) in the        region spanning positions 4664 to 4730 of the NS3 region as        shown in FIG. 6;    -   an HCV genomic sequence having a homology of more than 70%,        preferably more than 72%, most preferably more than 74% homology        to any of the sequences as represented in SEQ ID NO 29, 31, 33,        35, 37 or 39 (HCC153, HD10, BR36 sequences) in the region        spanning positions 4892 to 5292 in the NS3/NS4 region as shown        in FIG. 6 or 10;    -   an HCV genomic sequence of the BR36 subgroup of HCV type 3a as        having a homology of more than 95%, preferably 95,5%, most        preferably 96% homology to any of the sequences as represented        in SEQ ID NO 5, 7, 1, 3, 9 or 11 (BR34, BR33, BR36 sequences) in        the region spanning positions 8023 to 8235 of the NS5 region as        shown in FIG. 1;    -   an HCV genomic sequence of the BR36 subgroup of HCV type 3a as        having a homology of more than 96%, preferably 96.5%, most        preferably 97% homology to any of the sequences as represented        in SEQ ID NO 5, 7, 1, 3, 9 or 11 (BR34, BR33, BR36 sequences) in        the region spanning positions 8023 to 8192 of the NSSB region as        shown in FIG. 1;    -   an HCV genomic sequence of HCV type 3c being characterized as        having a homology of more than 79%, more preferably more than        81%, and most preferably more than 83% to the sequence as        represented in SEQ ID NO 149 (BE98 sequence) in the region        spanning positions 7932 to 8271 in the NS5B region as shown in        FIG. 1.

Preferentially the above-mentioned genomic HCV sequences depictsequences from the coding regions of all the above-mentioned sequences.

According to the nucleotide distance classification system (with saidnucleotide distances being calculated as explained above), saidsequences of said composition are selected from:

-   -   an HCV genomic sequence being characterized as having a        nucleotide distance of less than 0.44, preferably of less than        0.40, most preferably of less than 0.36 to any of the sequences        as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25 or 27 in        the region spanning positions 417 to 957 of the Core/E1 region        as shown in FIG. 4;    -   an HCV genomic sequence being characterized having a nucleotide        distance of less than 0.53, preferably less than 0.49, most        preferably of less than 0.45 to any of the sequences as        represented in SEQ ID NO 19, 21, 23, 25 or 27 in the region        spanning positions 574 to 957 of the E1 region as shown in FIG.        4;    -   an HCV genomic sequence characterized having a nucleotide        distance of less than 0.15, preferably less than 0.13, and most        preferably less than 0.11 to any of the sequences as represented        in SEQ ID NO 147 in the region spanning positions 1 to 378 of        the Core region as shown in FIG. 3;    -   an HCV genomic sequence of HVC type 3a being characterized as        having a nucleotide distance of less than 0.3, preferably less        than 0.26, most preferably of less than 0.22 to any of the        sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25        or 27 in the region spanning positions 417 to 957 in the Core/E1        region as shown in FIG. 4;    -   an HCV genomic sequence of HCV type 3a being characterized as        having a nucleotide distance of less than 0.35, preferably less        than 0.31, most preferably of less than 0.27 to any of the        sequences as represented in SEQ ID NO 13, 15, 17, 19, 21, 23, 25        or 27 in the region spanning positions 574 to 957 in the E1        region as shown in FIG. 4;    -   an HCV genomic sequence of the BR36 subgroup of HCV type 3a        being characterized as having a nucleotide sequence of less than        0.0423, preferably less than 0.042, preferably less than 0.0362        to any of the sequences as represented in SEQ ID NO 5, 7, 1, 3,        9 or 11 in the region spanning positions 8023 to 8235 of the NS5        region as shown in FIG. 1;    -   an HCV genomic sequence of HCV type 3c being characterized as        having a nucleotide distance of less than 0.255, preferably of        less than 0.25, more preferably of less than 0.21, most        preferably of less than 0.17 to the sequence as represented in        SEQ ID NO 149 in the region spanning positions 7932 to 8271 in        the NS5B region as shown in FIG. 1.

In the present application, the E1 sequences encoding the antigenicectodomain of the E1 protein, which does not overlap the carboxyterminalsignal-anchor sequences of E1 disclosed by Cha et al. (1992; WO92/19743), in addition to the NS4 epitope region, and a part of the NS5region are disclosed for 4 different isolates: BR33, BR34, BR36, HCC153and HD10, all belonging to type 3a (SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 35, 37 or 39).

Also within the present invention are new subtype 3c sequences (SEQ IDNO 147, 149 of the isolate BE98 in the Core and NS5 regions (see FIGS. 3and 1).

Finally the present invention also relates to a new subtype 3a sequenceas represented in SEQ ID NO 217 (see FIG. 1).

Also included within the present invention are sequence variants of thepolynucleic acids as selected from any of the nucleotide sequences asgiven in any of the above mentioned SEQ ID numbers, with said sequencevariants containing either deletions and/or insertions of one or morenucleotides, mainly at the extremities of oligonucleotides (either 3′ or5′), or substitutions of some non-essential nucleotides by others(including modified nucleotides an/or inosine), for example, a type 1 or2 sequence might be modified into a type 3 sequence by replacing somenucleotides of the type 1 or 2 sequence with type-specific nucleotidesof type 3 as shown in FIG. 1 (NS5 region), FIG. 3 (Core region), FIG. 4(Core/E1 region), FIG. 6 and 10 (NS3/NS4 region).

According to another embodiment, the present invention relates to apolynucleic acid composition as defined above, wherein said polynucleicacids correspond to a nucleotide sequence selected from any of thefollowing HCV type 5 genomic sequences:

-   -   an HCV genomic sequence as having a homology of more than 85%,        preferably more than 86%, most preferably more than 87% homology        to any of the sequences as represented in SEQ ID NO 41, 43, 45,        47, 49, 51, 53 (PC sequences) or 151 (BE95 sequence) in the        region spanning positions 1 to 573 of the Core region as shown        in FIG. 9 and 3;    -   an HCV genomic sequence as having a homology of more than 61%,        preferably more than 63%, more preferably more than 65%        homology, even more preferably more than 66% homology and most        preferably more than 67% homology (f.i. 69 and 71%) to any of        the sequences as represented in SEQ ID NO 41, 43, 45, 47, 49,        51, 53 (PC sequences), 153 or 155 (BE95, BE100 sequences) in the        region spanning positions 574 to 957 of the E1 region as shown        in FIG. 4;    -   an HCV genomic sequence having a homology of more than 76.5%,        preferably of more than 77%, most preferably of more than 78%        homology with any of the sequences as represented in SEQ ID NO        55, 57, 197 or 199 (PC sequences) in the region spanning        positions 3856 to 4209 of the NS3 region as shown in FIG. 6 or        10;    -   an HCV genomic sequence having a homology of more than 68%,        preferably of more than 70%, most preferably of more than 72%        homology with the sequence as represented in SEQ ID NO 157 (BE95        sequence) in the region spanning positions 980 to 1179 of the        E1/E2 region as shown in FIG. 13;    -   an HCV genomic sequence having a homology of more than 57%,        preferably more than 59%, most preferably more than 61% homology        to any of the sequences as represented in SEQ ID NO 59 or 61 (PC        sequences) in the region spanning positions 4936 to 5296 of the        NS4 region as shown in FIG. 6 or 10;    -   an HCV genomic sequence as having a homology of more than 93%,        preferably more than 93.5%, most preferably more than 94%        homology to any of the sequences as represented in SEQ ID NO 159        or 161 (BE95 or BE96 sequences) in the region spanning positions        7932 to 8271 of the NS5B region as shown in FIG. 1.

Preferentially the above-mentioned genomic HCV sequences depictsequences from the coding regions of all the above-mentioned sequences.

According to the nucleotide distance classification system (with saidnucleotide distances being calculated as explained above), saidsequences of said composition are selected from:

-   -   a nucleotide distance of less than 0.53, preferably less than        0.51, more preferably less than 0.49 for the E1 region to the        type 5 sequences depicted above;    -   a nucleotide distance of less than 0.3, preferably less than        0.28, more preferably of less than 0.26 for the Core region to        the type 5 sequences depicted above;    -   a nucleotide distance of less than 0.072, preferably less than        0.071, more preferably less than 0.070 for the NS5B region to        the type 5 sequences as depicted above.

Isolates with similar sequences in the 5′UR to a group of isolatesincluding SA1, SA3, and SA7 described in the 5′UR by Bukh et al. (1992),have been reported and described in the 5′UR and NS5 region as group Vby Cha et al. (1992; WO 92/19743). This group of isolates belongs totype 5a as described in the present invention (SEQ ID NO 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 151, 153, 155, 157, 159, 161, 197 and 199).

Also included within the present invention are sequence variants of thepolynucleic acids as selected from any of the nucleotide sequences asgiven in any of the above given SEQ ID numbers with said sequencevariants containing either deletion and/or insertions of one or morenucleotides, mainly at the extremities of oligonucleotides (either 3′ or5′), or substitutions of some non-essential nucleotides (i.e.nucleotides not essential to discriminate between different genotypes ofHCV) by others (including modified nucleotides an/or inosine), forexample, a type 1 or 2 sequence might be modified into a type 5 sequenceby replacing some nucleotides of the type 1 or 2 sequence withtype-specific nucleotides of type 5 as shown in FIG. 3 (Core region),FIG. 4 (Core/E1 region), FIG. 10 (NS3/NS4 region), FIG. 14 (E1/E2region).

Another group of isolates including BU74 and BU79 having similarsequences in the 5′UR to isolates including Z6 and Z7 as described inthe 5′UR by Bukh et al. (1992), have been described in the 5′UR andclassified as a new type 4 by the inventors of this application (Stuyveret al., 1993). Coding sequences, including core, E1 and NS5 sequences ofseveral new Gabonese isolates belonging to this group, are disclosed inthe present invention (SEQ ID NO 106, 108, 110, 112, 114, 116, 118, 120and 122).

According to yet another embodiment, the present invention relates to acomposition as defined above, wherein said polynucleic acids correspondto a nucleotide sequence selected from any of the following HCV type 4genomic sequences:

-   -   an HCV genomic sequence having a homology of more than 66%,        preferably more than 68%, most preferably more than 70% homology        in the E1 region spanning positions 574 to 957 to any of the        sequences as represented in SEQ ID NO 118, 120 or 122 (GB358,        GB549, GB809 sequences) as shown in FIG. 4;    -   an HCV genomic sequence having a homology of more than 71%,        preferably more than 72%, most preferably more than 74% homology        to any of the sequences as represented in SEQ ID NO 118, 120 or        122 (GB358, GB549, GB809 sequences) in the region spanning        positions 379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence having a homology of more than 92%,        preferably more than 93%, most preferably more than 94% homology        to any of the sequences as represented in SEQ ID NO 163 or 165        (GB809, CAM600 sequences) in the region spanning positions 1 to        378 of the Core/E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4c) having a homology of more        than 85%, preferably more than 86%, more preferably more than        86.5% homology, most preferably more than 87, more than 88 or        more than 89% homology to any of the sequences as represented in        SEQ ID NO 183, 185 or 187 (GB116, GB215, GB809 sequences) in the        region spanning positions 379 to 957 of the E1 region as shown        in FIG. 4;    -   an HCV genomic sequence (subtype 4a) having a homology of more        than 81%, preferably more than 83%, most preferably more than        85% homology to the sequence as represented in SEQ ID NO 189        (GB908 sequence) in the region spanning positions 379 to 957 of        the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4e) having a homology of more        than 85%, preferably more than 87%, most preferably more than        89% homology to any of the sequences as represented in SEQ ID NO        167 or 169 (CAM600, GB908 sequences) in the region spanning        positions 379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4f) having a homology of more        than 79%, preferably more than 81%, most preferably more than        83% homology to any of the sequences as represented in SEQ ID NO        171 or 173 (CAMG22, CAMG27 sequences) in the region spanning        positions 379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4g) having a homology of more        than 84%, preferably more than 86%, most preferably more than        88% homology to the sequence as represented in SEQ ID NO 175        (GB549 sequence) in the region spanning positions 379 to 957 of        the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4h) having a homology of more        than 83%, preferably more than 85%, most preferably more than        87% homology to the sequence as represented in SEQ ID NO 177        (GB438 sequence) in the region spanning positions 379 to 957 of        the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4i) as having a homology of        more than 76%, preferably more than 78%, most preferably more        than 80% homology to the sequence as represented in SEQ ID NO        179 (CAR4/1205 sequence) in the region spanning positions 379 to        957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4j?) having a homology of more        than 84%, preferably more than 86%, most preferably more than        88% homology to the sequence as represented in SEQ ID NO 181        (CAR4/901 sequence) in the region spanning positions 379 to 957        of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence as having a homology of more than 73%,        preferably more than 75%, most preferably more than 77% homology        to any of the sequences as represented in SEQ ID NO 106, 108,        110, 112, 114, or 116 (GB48, GB116, GB215, GB358, GB549, GB809        sequences) in the region spanning positions 7932 to 8271 of the        NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4c) having a homology of more        than 88%, preferably more than 89%, most preferably more than        90% homology to any of the sequences as represented in SEQ ID NO        106, 108, 110, or 112 (GB48, GB116, GB215, GB358 sequences) in        the region spanning positions 7932 to 8271 of the NS5 region as        shown in FIG. 1;    -   an HCV genomic sequence (subtype 4e) having a homology of more        than 88%, preferably more than 89%, most preferably more than        90% homology to any of the sequences as represented in SEQ ID NO        116 or 201 (GB809 or CAM 600 sequences) in the region spanning        positions 7932 to 8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4f) having a homology of more        than 87%, preferably more than 89%, most preferably more than        90% homology to the sequence as represented in SEQ ID NO 203        (CAMG22 sequence) in the region spanning positions 7932 to 8271        of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4g) as having a homology of        more than 85%, preferably more than 87%, most preferably more        than 89% homology to the sequence as represented in SEQ ID NO        114 (GB549 sequence) in the region spanning positions 7932 to        8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4h) as having a homology of        more than 86%, preferably more than 87%, more preferably more        than 88% homology, more preferably more than 89% homology to the        sequence as represented in SEQ ID NO 207 (GB437 sequence) in the        region spanning positions 7932 to 8271 of the NS5 region as        shown in FIG. 1;    -   an HCV genomic sequence (subtype 4i) having a homology of more        than 84%, preferably more than 86%, most preferably more than        88% homology to the sequence as represented in SEQ ID NO 209        (CAR4/1205 sequence) in the region spanning positions 7932 to        8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4j) having a homology of more        than 81%, preferably more than 83%, most preferably more than        85% homology to the sequence as represented in SEQ ID NO 211        (CAR1/501 sequence) in the region spanning positions 7932 to        8271 of the NS5 region as shown in FIG. 1.    -   Preferentially the above-mentioned genomic HCV sequences depict        sequences from the coding regions of all the above-mentioned        sequences.

According to the nucleotide distance classification system (with saidnucleotide distances being calculated as explained above), saidsequences of said composition are selected from:

-   -   an HCV genomic sequence (type 4) being characterized as having a        nucleotide distance of less than 0.52, 0.50, 0.4880, 0.46, 0.44,        0.43 or most preferably less than 0.42 in the region spanning        positions 574 to 957 to any of the sequences as represented in        SEQ ID NO 118, 120 or 122 in the region spanning positions 1 to        957 of the Core/E1 region as shown in FIG. 4;    -   an HCV genomic sequence (type 4) being characterized as having a        nucleotide distance of less than 0.39, 0.36 0.34 0.32 or most        preferably less than 0.31 to any of the sequences as represented        in SEQ ID NO 118, 120 or 122 in the region spanning positions        379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4c) being characterized as        having a nucleotide distance of less than 0.27, 0.26, 0.24,        0.22, 0.20, 0.18, 0.17, 0.162, 0.16 or most preferably less than        0.15 to any of the sequences as represented in SEQ ID NO 183,        185 or 187 in the region spanning positions 379 to 957 of the E1        region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4a) being characterized as        having a nucleotide distance of less than 0.30, 0.28, 0.26,        0.24, 0.22, 0.21 or most preferably of less than 0.205 to the        sequence as represented in SEQ ID NO 189 in the region spanning        positions 379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4e) being characterized as        having a nucleotide distance of less than 0.26, 0.25, 0.23,        0.21, 0.19, 0.17, 0.165, most preferably less than 0.16 to any        of the sequences as represented in SEQ ID NO 167 or 169 in the        region spanning positions 379 to 957 of the E1 region as shown        in FIG. 4;    -   an HCV genomic sequence (subtype 4f) being characterized as        having a nucleotide distance of less than 0.26, 0.24, 0.22,        0.20, 0.18, 0.16, 0.15 or most preferably less than 0.14 to any        of the sequences as represented in SEQ ID NO 171 or 173 in the        region spanning positions 379 to 957 of the E1 region as shown        in FIG. 4;    -   an HCV genomic sequence (subtype 4g) being characterized as        having a nucleotide distance of less than 0.20, 0.19, 0.18, 0.17        or most preferably of less than 0.16 to the sequence as        represented in SEQ ID NO 175 in the region spanning positions        379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4h) being characterized as        having a nucleotide distance of less than 0.20, 0.19, 0.18, 0.17        and most preferably of less than 0.16 to the sequence as        represented in SEQ ID NO 177 in the region spanning positions        379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4i) being characterized as        having a nucleotide distance of less than 0.27, 0.25, 0.23, 0.21        and preferably less than 0.16 to the sequence as represented in        SEQ ID NO 179 in the region spanning positions 379 to 957 of the        E1 region as shown in FIG. 4;    -   an HCV genomic sequence (subtype 4j?) being characterized as        having a nucleotide distance of less than 0.19, 0.18, 0.17,        0.165 and most preferably of less than 0.16 to the sequence as        represented in SEQ ID NO 181 in the region spanning positions        379 to 957 of the E1 region as shown in FIG. 4;    -   an HCV genomic sequence (type 4) being characterized as having a        nucleotide distance of less than 0.35, 0.34, 0.32 and most        preferably of less than 0.30 to any of the sequences as        represented in SEQ ID NO 106, 108, 110, 112, 114, or 116 in the        region spanning positions 7932 to 8271 of the NS5 region as        shown in FIG. 1;    -   an HCV genomic sequence (subtype 4c) being characterized as        having a nucleotide distance of less than 0.18, 0.16, 0.14,        0.135, 0.13, 0.1275 or most preferably less than 0.125 to any of        the sequences as represented in SEQ ID NO 106, 108, 110, or 112        in the region spanning positions 7932 to 8271 of the NS5 region        as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4e) being characterized as        having a nucleotide distance of less than 0.15, 0.14, 0.135,        0.13 and most preferably of less than 0.125 to any of the        sequences as represented in SEQ ID NO 116 or 201 in the region        spanning positions 7932 to 8271 of the NS5 region as shown in        FIG. 1;    -   an HCV genomic sequence (subtype 4f) being characterized as        having a nucleotide distance of less than 0.15, 0.14, 0.135,        0.13 or most preferably less than 0.125 to the sequence as        represented in SEQ ID NO 203 in the region spanning positions        7932 to 8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4g) being characterized as        having a nucleotide distance of less than 0.17, 0.16, 0.15,        0.14, 0.13 or most preferably less than 0.125 to the sequence as        represented in SEQ ID NO 114 in the region spanning positions        7932 to 8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4h) being characterized as        having a nucleotide distance of less than 0.155, 0.15, 0.145,        0.14, 0.135, 0.13 or most preferably less than 0.125 to the        sequence as represented in SEQ ID NO 207 in the region spanning        positions 7932 to 8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4i) being characterized as        having a nucleotide distance of less than 0.17, 0.16, 0.15,        0.14, 0.13 or most preferably of less than 0.125 to the sequence        as represented in SEQ ID NO 209 in the region spanning positions        7932 to 8271 of the NS5 region as shown in FIG. 1;    -   an HCV genomic sequence (subtype 4j) being characterized as        having a nucleotide distance of less than 0.21, 0.20, 0.19,        0.18, 0.17, 0.16, 0.15, 0.14, 0.13 and most preferably of less        than 0.125 to the sequence as represented in SEQ ID NO 211 in        the region spanning positions 7932 to 8271 of the NS5 region as        shown in FIG. 1.

Also included within the present invention are sequence variants of thepolynucleic acids as selected from any of the nucleotide sequences asgiven in any of the above given SEQ ID numbers with said sequencevariants containing either deletion and/or insertions of one or morenucleotides, mainly at the extremities of oligonucleotides (either 3′ or5′), or substitutions of some non-essential nucleotides (i.e.nucleotides not essential to discriminate between different genotypes ofHCV) by others (including modified nucleotides an/or inosine), forexample, a type 1 or 2 sequence might be modified into a type 4 sequenceby replacing some nucleotides of the type 1 or 2 sequence withtype-specific nucleotides of type 4 as shown in FIG. 3 (Core region),FIG. 4 (Core/E1 region), FIG. 10 (NS3/NS4 region), FIG. 14 (E1/E2region).

The present invention also relates to a sequence as represented in SEQID NO 193 (GB724 sequence).

After aligning NS5 or E1 sequences of GB48, GB, 116, GB215, GB358, GB549and GB809, these isolates clearly segregated into 3 subtypes within type4: GB48, GB116, GB215 and GB358 belong to the sybtype designated 4c,GB549 to subtype 4g and GB809 to subtype 4e. In NS5, GB809 (subtype 4e)showed a higher nucleic acids homology to subtype 4c isolates(85.6–86.8%) than to GB549 (subtype 4g, 79.7%), while GB549 showedsimilar homologies to both other subtypes (78.8 to 80% to subtype 4c and79.7% to subtype 4e). In E1, subtype 4c showed equal nucleic acidhomologies of 75.2% to subtypes 4g and 4e while 4g and 4e were 78.4%homologous. At the amino acid level however, subtype 4e showed a normalhomology to subtype 4c (80.2%), while subtype 4g was more homologous to4c (83.3%) and 4e (84.1%).

According to yet another embodiment, the present invention relates to acomposition as defined above, wherein said polynucleic acids correspondto a nucleotide sequence selected from any of the following HCV type 2dgenomic sequences:

-   -   an HCV genomic sequence as having a homology of more than 78%,        preferably more than 80%, most preferably more than 82% homology        to the sequence as represented in SEQ ID NO (NE92) 143 in the        region spanning positions 379 to 957 of the Core/E1 region as        shown in FIG. 4;    -   an HCV genomic sequence as having a homology of more than 74%,        preferably more than 76%, most preferably more than 78% homology        to the sequence as represented in SEQ ID NO 143 (NE92) in the        region spanning positions 574 to 957 as shown in FIG. 4;    -   an HCV genomic sequence as having a homology of more than 87%,        preferably more than 89%, most preferably more than 91% homology        to the sequence as represented in SEQ ID NO 145 (NE92) in the        region spanning positions 7932 to 8271 of the NS5B region as        shown in FIG. 1.

Preferentially the above-mentioned genomic HCV sequences depictsequences from the coding regions of all the above-mentioned sequences.

According to the nucleotide distance classification system (with saidnucleotide distances being calculated as explained above), saidsequences of said composition are selected from:

-   -   a nucleotide distance of less than 0.32, preferably less than        0.31, more preferably less than 0.30 for the E1 region (574        to 957) to any of the above specified sequences;    -   a nucleotide distance of less than 0.08, preferably less than        0.07, more preferably less than 0.06 for the Core region (1        to 378) to any of the above given sequences    -   a nucleotide distance of less than 0.15, preferantially less        than 0.13, more preferentially less than 0.12 for the NS5B        region to any of the above-specified sequences.

Polynucleic acid sequences according to the present invention which arehomologous to the sequences as represented by a SEQ ID NO can becharacterized and isolated according to any of the techniques known inthe art, such as amplification by means of type or subtype specificprimers, hybridization with type or subtype specific probes under moreor less stringent conditions, serological screening methods (seeexamples 4 and 11) or via the LiPA typing system.

Polynucleic acid sequences of the genomes indicated above from regionsnot yet depicted in the present examples, figures and sequence listingcan be obtained by any of the techniques known in the art, such asamplification techniques using suitable primers from the type or subtypespecific sequences of the present invention.

The present invention relates also to a composition as defined above,wherein said polynucleic acid is liable to act as a primer foramplifying the nucleic acid of a certain isolate belonging to thegenotype from which the primer is derived.

An example of a primer according to this embodiment of the invention isHCPr 152 as shown in table 7 (SEQ ID NO 79).

The term “primer” refers to a single stranded DNA oligonucleotidesequence capable of acting as a point of initiation for synthesis of aprimer extension product which is complementary to the nucleic acidstrand to be copied. The length and the sequence of the primer must besuch that they allow to prime the synthesis of the extension products.Preferably the primer is about 5–50 nucleotides. Specific length andsequence will depend on the complexity of the required DNA or RNAtargets, as well as on the conditions of primer use such as temperatureand ionic strength.

The fact that amplification primers do not have to match exactly withcorresponding template sequence to warrant proper amplification is amplydocumented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction(PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al.,1988; Wu & Wallace, 1989; Barany, 1991), nucleic acid sequence-basedamplification (NASBA; Guatelli et al., 1990; Compton, 1991),transcription-based amplification system (TAS; Kwoh et al., 1989),strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992)or amplification by means of QB replicase (Lizardi et al., 1988; Lomeliet al., 1989) or any other suitable method to amplify nucleic acidmolecules using primer extension. During amplification, the amplifiedproducts can be conveniently labelled either using labelled primers orby incorporating labelled nucleotides. Labels may be isotopic (³²P, ³⁵S,etc.) or non-isotopic (biotin, digoxigenin, etc.). The amplificationreaction is repeated between 20 and 80 times, advantageously between 30and 50 times.

The present invention also relates to a composition as defined above,wherein said polynucleic acid is able to act as a hybridization probefor specific detection and/or classification into types of a nucleicacid containing said nucleotide sequence, with said oligonucleotidebeing possibly labelled or attached to a solid substrate.

The term “probe” refers to single stranded sequence-specificoligonucleotides which have a sequence which is complementary to thetarget sequence of the HCV genotype(s) to be detected.

Preferably, these probes are about 5 to 50 nucleotides long, morepreferably from about 10 to 25 nucleotides.

The term “solid support” can refer to any substrate to which anoligonucleotide probe can be coupled, provided that it retains itshybridization characteristics and provided that the background level ofhybridization remains low. Usually the solid substrate will be amicrotiter plate, a membrane (e.g. nylon or nitrocellulose) or amicrosphere (bead). Prior to application to the membrane or fixation itmay be convenient to modify the nucleic acid probe in order tofacilitate fixation or improve the hybridization efficiency. Suchmodifications may encompass homopolymer tailing, coupling with differentreactive groups such as aliphatic groups, NH₂ groups, SH groups,carboxylic groups, or coupling with biotin or haptens.

The present invention also relates to the use of a composition asdefined above for detecting the presence of one or more HCV genotypes,more particularly for detecting the presence of a nucleic acid of any ofthe HCV genotypes having a nucleotide sequence as defined above, presentin a biological sample liable to contain them, comprising at least thefollowing steps:

-   (i) possibly extracting sample nucleic acid,-   (ii) possibly amplifying the nucleic acid with at least one of the    primers as defined above or any other HCV subtype 2d, HCV type 3,    HCV type 4, HCV type 5 or universal HCV primer,-   (iii) hybrizing the nucleic acids of the biological sample, possibly    under denatured conditions, and with said nucleic acids being    possibly labelled during or after amplification, at appropriate    conditions with one or more probes as defined above, with said    probes being preferably attached to a solid substrate,-   (iv) washing at appropriate conditions,-   (v) detecting the hybrids formed,-   (vi) inferring the presence of one or more HCV genotypes present    from the observed hybridization pattern.

Preferably, this technique could be performed in the Core or NS5Bregion.

The term “nucleic acid” can also be referred to as analyte strand andcorresponds to a single- or double-stranded nucleic acid molecule. Thisanalyte strand is preferentially positive- or negative stranded RNA,cDNA or amplified cDNA.

The term “biological sample” refers to any biological sample (tissue orfluid) containing HCV nucleic acid sequences and refers moreparticularly to blood serum or plasma samples.

The term “HCV subtype 2d primer” refers to a primer which specificallyamplifies HCV subtype 2d sequences present in a sample (see Examplessection and figures).

The term “HCV type 3 primer” refers to a primer which specificallyamplifies HCV type 3 sequences present in a sample (see Examples sectionand figures).

The term “HCV type 4 primer” refers to a primer which specificallyamplifies HCV type 4 genomes present in a sample.

The term “universal HCV primer” refers to oligonucleotide sequencescomplementary to any of the conserved regions of the HCV genome.

The term “HCV type 5 primer” refers to a primer which specificallyamplifies HCV type 5 genomes present in a sample. The term “universalHCV primer” refers to oligonucleotide sequences complementary to any ofthe conserved regions of the HCV genome.

The expression “appropriate” hybridization and washing conditions are tobe understood as stringent and are generally known in the art (e.g.Maniatis et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory, 1982).

However, according to the hybridization solution (SSC, SSPE, etc.),these probes should be hybridized at their appropriate temperature inorder to attain sufficient specificity.

The term “labelled” refers to the use of labelled nucleic acids. Thismay include the use of labelled nucleotides incorporated during thepolymerase step of the amplification such as illustrated by Saiki et al.(1988) or Bej et al. (1990) or labelled primers, or by any other methodknown to the person skilled in the art.

The process of the invention comprises the steps of contacting any ofthe probes as defined above, with one of the following elements:

-   -   either a biological sample in which the nucleic acids are made        available for hybridization,    -   or the purified nucleic acids contained in the biological sample    -   or a single copy derived from the purified nucleic acids,    -   or an amplified copy derived from the purified nucleic acids,        with said elements or with said probes being attached to a solid        substrate.

The expression “inferring the presence of one or more HCV genotypespresent from the observed hybridization pattern” refers to theidentification of the presence of HCV genomes in the sample by analyzingthe pattern of binding of a panel of oligonucleotide probes. Singleprobes may provide useful information concerning the presence or absenceof HCV genomes in a sample. On the other hand, the variation of the HCVgenomes is dispersed in nature, so rarely is any one probe able toidentify uniquely a specific HCV genome. Rather, the identity of an HCVgenotype may be inferred from the pattern of binding of a panel ofoligonucleotide probes, which are specific for (different) segments ofthe different HCV genomes. Depending on the choice of theseoligonucleotide probes, each known HCV genotype will correspond to aspecific hybridization pattern upon use of a specific combination ofprobes. Each HCV genotype will also be able to be discriminated from anyother HCV genotype amplified with the same primers depending on thechoice of the oligonucleotide probes. Comparison of the generatedpattern of positively hybridizing probes for a sample containing one ormore unkown HCV sequences to a scheme of expected hybridizationpatterns, allows one to clearly infer the HCV genotypes present in saidsample. The present invention thus relates to a method as defined above,wherein one or more hybridization probes are selected from any of SEQ IDNO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 or 61, 106, 108, 110,112, 114, 116, 118, 120, 122, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 198, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 222, 269 or sequence variants thereof, with said sequencevariants containing deletions and/or insertions of one or morenucleotides, mainly at their extremities (either 3′ or 5′), orsubstitutions of some non-essential nucleotides (i.e. nucleotides notessential to discriminate between genotypes) by others (includingmodified nucleotides or inosine), or with said variants consisting ofthe complement of any of the above-mentioned oligonucleotide probes, orwith said variants consisting of ribonucleotides instead ofdeoxyribonucleotides, all provided that said variant probes can becaused to hybridize with the same specificity as the oligonucleotideprobes from which they are derived.

In order to distinguish the amplified HCV genomes from each other, thetarget polynucleic acids are hybridized to a set of sequence-specificDNA probes targetting HCV genotypic regions located in the HCVpolynucleic acids.

Most of these probes target the most type-specific regions of HCVgenotypes, but some can be caused to hybridize to more than one HCVgenotype. According to the hybridization solution (SSC, SSPE, etc.),these probes should be stringently hybridized at their appropriatetemperature in order to attain sufficient specificity. However, byslightly modifying the DNA probes, either by adding or deleting one or afew nucleotides at their extremities (either 3′ or 5′), or substitutingsome non-essential nucleotides (i.e. nucleotides not essential todiscriminate between types) by others (including modified nucleotides orinosine) these probes or variants thereof can be caused to hybridizespecifically at the same hybridization conditions (i.e. the sametemperature and the same hybridization solution). Also changing theamount (concentration) of probe used may be beneficial to obtain morespecific hybridization results. It should be noted in this context, thatprobes of the same length, regardless of their GC content, willhybridize specifically at approximately the same temperature in TMAClsolutions (Jacobs et al., 1988).

Suitable assay methods for purposes of the present invention to detecthybrids formed between the oligonucleotide probes and the nucleic acidsequences in a sample may comprise any of the assay formats known in theart, such as the conventional dot-blot format, sandwich hybridization orreverse hybridization. For example, the detection can be accomplishedusing a dot blot format, the unlabelled amplified sample being bound toa membrane, the membrane being incorporated with at least one labelledprobe under suitable hybridization and wash conditions, and the presenceof bound probe being monitored.

An alternative and preferred method is a “reverse” dot-blot format, inwhich the amplified sequence contains a label. In this format, theunlabelled oligonucleotide probes are bound to a solid support andexposed to the labelled sample under appropriate stringent hybridizationand subsequent washing conditions. It is to be understood that also anyother assay method which relies on the formation of a hybrid between thenucleic acids of the sample and the oligonucleotide probes according tothe present invention may be used.

According to an advantageous embodiment, the process of detecting one ormore HCV genotypes contained in a biological sample comprises the stepsof contacting amplified HCV nucleic acid copies derived from thebiological sample, with oligonucleotide probes which have beenimmobilized as parallel lines on a solid support.

According to this advantageous method, the probes are immobilized in aLine Probe Assay (LiPA) format. This is a reverse hybridization format(Saiki et al., 1989) using membrane strips onto which severaloligonucleotide probes (including negative or positive controloligonucleotides) can be conveniently applied as parallel lines.

The invention thus also relates to a solid support, preferably amembrane strip, carrying on its surface, one or more probes as definedabove, coupled to the support in the form of parallel lines.

The LiPA is a very rapid and user-friendly hybridization test. Resultscan be read 4 h. after the start of the amplification. Afteramplification during which usually a non-isotopic label is incorporatedin the amplified product, and alkaline denaturation, the amplifiedproduct is contacted with the probes on the membrane and thehybridization is carried out for about 1 to 1,5 h hybridized polynucleicacid is detected. From the hybridization pattern generated, the HCV typecan be deduced either visually, but preferably using dedicated software.The LiPA format is completely compatible with commercially availablescanning devices, thus rendering automatic interpretation of the resultsvery reliable. All those advantages make the LiPA format liable for theuse of HCV detection in a routine setting. The LiPA format should beparticularly advantageous for detecting the presence of different HCVgenotypes.

The present invention also relates to a method for detecting andidentifying novel HCV genotypes, different from the known HCV genomes,comprising the steps of:

-   -   determining to which HCV genotype the nucleotides present in a        biological sample belong, according to the process as defined        above,    -   in the case of observing a sample which does not generate a        hybridization pattern compatible with those defined in Table 3,        sequencing the portion of the HCV genome sequence corresponding        to the aberrantly hybridizing probe of the new HCV genotype to        be determined.

The present invention also relates to the use of a composition asdefined above, for detecting one or more genotypes of HCV present in abiological sample liable to contain them, comprising the steps of:

-   -   (i) possibly extracting sample nucleic acid,    -   (ii) amplifying the nucleic acid with at least one of the        primers as defined above,    -   (iii) sequencing the amplified products    -   (iv) inferring the HCV genotypes present from the determined        sequences by comparison to all known HCV sequences.

The present invention also relates to a composition consisting of orcomprising at least one peptide or polypeptide comprising a contiguoussequence of at least 5 amino acids corresponding to a contiguous aminoacid sequence encoded by at least one of the HCV genomic sequences asdefined above, having at least one amino acid differing from thecorresponding region of known HCV (type 1 and/or type 2 and/or type 3)polyprotein sequences as shown in Table 3, or muteins thereof.

It is to be noted that, at the level of the amino acid sequence, anamino acid difference (with respect to known HCV amino acid sequences)is necessary, which means that the polypeptides of the inventioncorrespond to polynucleic acids having a nucleotide difference (withknown HCV polynucleic acid sequences) involving an amino aciddifference.

The new amino acid sequences, as deduced from the disclosed nucleotidesequences (see SEQ ID NO 1 to 62 and 106 to 123 and 143 to 218, 223 and270), show homologies of only 59.9 to 78% with prototype sequences oftype 1 and 2 for the NS4 region, and of only 53.9 to 68.8% withprototype sequences of type 1 and 2 for the E1 region. As the NS4 regionis known to contain several epitopes, for example characterized inpatent application EP-A-0 489 968, and as the E1 protein is expected tobe subject to immune attack as part of the viral envelope and expectedto contain epitopes, the NS4 and E1 epitopes of the new type 3, 4 and 5isolates will consistently differ from the epitopes present in type 1and 2 isolates. This is examplified by the type-specificity of NS4synthetic peptides as presented in example 4, and the type-specificityof recombinant E1 proteins in example 11.

After aligning the new subtype 2d, type 3, 4 and 5 (see SEQ ID NO 1 to62 and 106 to 123 and 143 to 218, 223 and 270) amino acid sequences withthe prototype sequences of type 1a, 1b, 2a, and 2b, type- andsubtype-specific variable regions can be delineated as presented inFIGS. 5 and 7.

As to the muteins derived from the polypeptides of the invention, Table4 gives an overview of the amino acid substitutions which could be thebasis of some of the muteins as defined above.

The peptides according to the present invention contain preferably atleast 5 contiguous HCV amino acids, preferably however at least 8contiguous amino acids, at least 10 or at least 15 (for instance atleast 9, 11, 12, 13, 14, 20 or 25 amino acids) of the new HCV sequencesof the invention.

TABLE 4 Amino acids Synonymous groups Ser (S) Ser, Thr, Gly, Asn Arg (R)Arg, His, Lys, Glu, Gln Leu (L) Leu; Ile, Met, Phe, Val, Tyr Pro (P)Pro, Ala, Thr, Gly Thr (T) Thr, Pro, Ser, Ala, Gly, His, Gln Ala (A)Ala, Pro, Gly, Thr Val (V) Val, Met, Ile, Tyr, Phe, Leu, Val Gly (G)Gly, Ala, Thr, Pro, Ser Ile (I) Ile, Met, Leu, Phe, Val, Ile, Tyr Phe(F) Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr (Y) Tyr, Phe, Trp, Met, Ile,Val, Leu Cys (C) Cys, Ser, Thr, Met His (H) His, Gln, Arg, Lys, Glu, ThrGln (Q) Gln, Glu, His, Lys, Asn, Thr, Arg Asn (N) Asn, Asp, Ser, Gln Lys(K) Lys, Arg, Glu, Gln, His Asp (D) Asp, Asn, Glu, Gln Glu (E) Glu, Gln,Asp, Lys, Asn, His, Arg Met (M) Met, Ile, Leu, Phe, Val

The polypeptides of the invention, and particularly the fragments, canbe prepared by classical chemical synthesis.

The synthesis can be carried out in homogeneous solution or in solidphase.

For instance, the synthesis technique in homogeneous solution which canbe used is the one described by Houbenweyl in the book entitled “Methodeder organischen chemie” (Method of organic chemistry) edited by E.Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.

The polypeptides of the invention can also be prepared in solid phaseaccording to the methods described by Atherton and Shepard in their bookentitled “Solid phase peptide synthesis” (IRL Press, Oxford, 1989).

The polypeptides according to this invention can be prepared by means ofrecombinant DNA techniques as described by Maniatis et al., MolecularCloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory,1982).

The present invention relates particularly to a polypeptide or peptidecomposition as defined above, wherein said contiguous sequence containsin its sequence at least one of the following amino acid residues: L7,Q43, M44, S60, R67, Q70, T71, A79, A87, N106, K115, A127, A190, S130,V134, G142, I144, E152, A157, V158, P165, S177 or Y177, I178, V180 orE180 or F182, R184, I186, H187, T189, A190, S191 or G191, Q192 or L192or I192 or V192 or E192, N193 or H193 or P193, W194 or Y194, H195, A197or I197 or V197 or T197, V202, I203 or L203, Q208, A210, V212, F214,T216, R217 or D217 or E217 or V217, H218 or N218, H219 or V219 or L219,L227 or I227, M231 or E231 or Q231, T232 or D232 or A232 or K232, Q235or I235, A237 or T237, I242, I246, S247, S248, V249, S250 or Y250, I251,or V251 or M251 or F251, D252, T254 or V254, L255 or V255, E256 or A256,M258 or F258 or V258, A260 or Q260 or S260, A261, T264 or Y264, M265,I266 or A266, A267, G268 or T268, F271 or M271 or V271, I277, M280 orH280, I284 or A284 or L84, V274, V291, N292 or S292, R293 or I293 orY293, Q294 or R94, L297 or I297 or Q297, A299 or K299 or Q299, N303 orT303, T308 or L308, T310 or F310 or A310 or D310 or V310, L313, G317 orQ317, L333, S351, A358, A359, A363, S364, A366, T369, L373, F376, Q386,I387, S392, I399, F402, I403, R405, D454, A461, A463, T464, K484, Q500,E501, S521, K522, H524, N528, S531, S532, V534, F536, F537, M539, I546,C1282, A1283, H1310, V1312, Q1321, P1368, V1372, V1373, K1405, Q1406,S1409, A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529,I1543, N1567, D1556, N1567, M1572, Q1579, L1581, S1583, F1585, V1595,E1606 or T1606, M1611, V1612 or L1612, P1630, C1636, P1651, T1656 orI1656, L1663, V1667, V1677, A1681, H1685, E1687, G1689, V1695, A1700,Q1704, Y1705, A1713, A1714 or S1714, M1718, D1719, A1721 or T1721,R1722, A1723 or V1723, H1726 or G1726, E1730, V1732, F1735, I1736,S1737, R1738, T1739, G1740, Q1741, K1742, Q1743, A1744, T1745, L1746,E1747 or K1747, I1749, A1750, T1751 or A1751, V1753, N1755, K1756,A1757, P1758, A1759, H1762, T1763, Y1764, P2645, A2647, K2650, K2653 orL2653, S2664, N2673, F2680, K2681, L2686, H2692, Q2695 or L2695 or12695, V2712, F2715, V2719 or Q2719, T2722, T2724, S2725, R2726, G2729,Y2735, H2739, I2748, G2746 or I2746, I2748, P2752 or K2752, P2754 orT2754, T2757 or P2757, with said notation being composed of a letterrepresenting the amino acid residue by its one-letter code, and a numberrepresenting the amino acid numbering according to Kato et al., 1990 asshown in Table 1 (comparison with other isolates). See also thenumbering in FIGS. 2, 5, 7, and 11 (alignment amino acid sequences).

Within the group of unique and new amino acid residues of the presentinvention, the following residues were found to be specific for thefollowing types of HCV according to the HCV classification system usedin the present invention:

-   -   Q208, R217, E231, I235, I246, T264, I266, A267, F271, K299,        L2686, Q2719 which are specific for the HCV subtype 2d sequences        of the present invention as shown in FIGS. 5 and 2;

    -   Q43, S60, R67, F182, I186, H187, A190, S191, L192, W194, V202,        L203, V219, Q231, D232, A237, T-254, M280, Q299, T303, L308,        and/or L313 which are specific for the Core/E1 region of HCV        type 3 of the invention as shown in FIG. 5;

    -   D1556, Q1579, L1581, S1584, F1585, E1606, V1612, P1630, C1636,        T1656, L1663, H1685, E1687, G1689, V1695, Y1705, A1713, A1714,        A1721, V1723, H1726, R1738, Q1743, A1744, E1747, 11749, A1751,        A1759 and/or H1762 which are specific for the NS3/4 region of        HCV type 3 sequences of the invention as shown in FIG. 7;

    -   K2665, D2666, R2670 which are specific for the NS5B region of        HCV type 3 of the invention as shown in FIG. 2;

    -   L7, A79, A127, S130, E152, V158, S177 or Y177, V180 or E180,        R184, T189, Q192 or E192 or I192, N193 or H193, I197 or V197,        I203, A210, V212, E217, H218, H219, L227, A232, V249, I251 or        M251, D252, L255 or V255, E256, M258 or V258 or F258, A260 or        Q260, M265, T268, V271, V274, M280, I284, N292 or S292, Q294,        L297 or I297, T308, A310 or D310 or V310 or T310, and G317 which        are specific for the core/E1 region of HCV type 4 sequences of        the present invention as shown in FIG. 5;

    -   P2645, K2650, K2653, G2656, V2658, T2668, N2673 or N2673, K2681,        H2686, D2691, L2692, Q2695 or L2695 or I2695, Y2704, V2712,        F2715, V2719, I2722, S2725, G2729, Y2735, G2746 or I2746, P2752        or K2752, Q2753, P2754 or T2754, T2757 or P2757 which are        specific for the NS5B region of the HCV type 4 sequences of the        present invention as shown in FIG. 2;

    -   M44, Q70, A87, N106, K115, V137, G142, P165, I178, F251, A299,        N303, Q317 which are specific for the Core/E1 region of the HCV        type 5 sequence of the present invention as shown in FIG. 5;

    -   L333, S351, A358, A359, A363, S364, A366, T369, L373, F376,        Q386, I387, S392, I399, F102, I403, R405, D454, A461, A463,        T464, K484, Q500, E501, S521, K522, H524, N528, S532, V534,        F537, M539, I546 which are specific for the E1/E2 region of the        HCV type 5 sequences of the present invention as shown in FIG.        12;

    -   C1282, A1283, V1312, Q1321, P1368, V1372, K1405, Q1406, S1409,        A1424, A1429, C1435, S1436, S1456, H1496, A1504, D1510, D1529,        I1543, N1567, M1572, V1595, T1606, M1611, L1612, I1656, V1667,        A1681, A1700, A1713, S1714, M1718, D1719, T1721, R1722, A1723,        G1726, F1735, I1736, S1737, T1739, G1740, K1742, T1745, L1746,        K1747, A1750, V1753, N1755, A1757, D1758, T1763, and Y1764 which        are specific for the NS3/NS4 region of HCV type 5 sequences of        the invention as shown in FIG. 7;

    -   

A2647, L2653, S2674, F2680, T2724, R2726, Y2730, H2739 which arespecific for the NS5B region of the HCV type 5 sequences of the presentinvention as shown in FIG. 2;

-   -   A256, P1631, V1677, Q1704, E1730, V1732, Q1741 and T1751 which        are specific for the HCV type 3 and 5 sequences of the present        invention as shown in FIG. 5 and 7;    -   T71, A157, I227, T237, T240, Y250, V251, S260, M271, T2673,        T2722, I2748 which are specific for the HCV type 3 and 4        sequences of the present invention as shown in FIGS. 5 and 2,    -   V192, Y194, A197, P249, S250, R294 which are specific for the        HCV type 4 and 5 sequences of the present invention as shown in        FIG. 5;    -   I293 which is specific for the HCV type 4 and subtype 2d        sequence of the present invention as shown in FIG. 5;    -   D217 and R294 which are specific for the HCV type 3, 4 and 5        sequences of the present invention as shown in FIG. 5;    -   L192 which is specific for the HCV type 3 and subtype 2d        sequences of the present invention as shown in FIG. 5;    -   G191 and T197 which are specific for the HCV type 3, 4 and        subtype 2d sequences of the present invention as shown in FIG.        5;    -   K232 which is specific for the HCV subtype 2d en type 5        sequences of the present invention as shown in FIG. 5.        and with said notation being composed of a letter, unambiguously        representing the amino acid by its one-letter code, and a number        representing the amino acid numbering according to Kato et al.,        1990 (see also Table 1 for comparison with other isolates), as        well as FIG. 2 (NS5 region), FIG. 5 (Core/E1 region), FIG. 7        (NS3/NS4 region), FIG. 12 (E1/E2 region). Some of the        above-mentioned amino acids may be contained in type or subtype        specific epitopes.

For example M231 (detected in type 5) refers to a methionine at position231. A glutamine (Q) is present at the same position 231 in type 3isolates, whereas this position is occupied by an arginine in type 1isolates and by a lysine (K) or asparagine (N) in type 2 isolates (seeFIG. 5).

The peptide or polypeptide according to this embodiment of the inventionmay be possibly labelled, or attached to a solid substrate, or coupledto a carrier molecule such as biotin, or mixed with a proper adjuvant.

The variable region in the core protein (V-CORE in FIG. 5) has beenshown to be useful for serotyping (Machida et al., 1992). The sequenceof the disclosed type 5 sequence in this region shows type-specificfeatures. The peptide from amino acid 70 to 78 shows the followingunique sequence for the sequences of the present inevntion (see FIG. 5):

QPTGRSWGQ (SEQ ID NO 93) RSEGRTSWAQ (SEQ ID NO 220) and RTEGRTSWAQ (SEQID NO 221)

Another preferred V-Core spanning region is the peptide spanningpositions 60 to 78 of subtype 3c with sequence:

SRRQPIPRARRTEGRSWAQ (SEQ ID NO 268)

Five type-specific variable regions (V1 to V5) can be identified afteraligning E1 amino acid sequences of the 4 genotypes, as shown in FIG. 5.

Region V1 encompasses amino acids 192 to 203, this is the amino-terminal10 amino acids of the E1 protein. The following unique sequences asshown in FIG. 5 can be deduced:

LEWRNTSGLYVL (SEQ ID NO 83) VNYRNASGIYHI (SEQ ID NO 126) QHYRNISGIYHV(SEQ ID NO 127) EHYRNASGIYHI (SEQ ID NO 128) IHYRNASGIYHI (SEQ ID NO224) VPYRNASGIYHV (SEQ ID NO 84) VNYRNASGIYHI (SEQ ID NO 225)VNYRNASGVYHI (SEQ ID NO 226) VNYHNTSGIYHL (SEQ ID NO 227) QHYRNASGIYHV(SEQ ID NO 228) QHYRNVSGIYHV (SEQ ID NO 229) IHYRNASDGYYI (SEQ ID NO230) LQVKNTSSSYMV (SEQ ID NO 231)

Region V2 encompasses amino acids 213 to 223. The following uniquesequences can be found in the V2 region as shown in FIG. 5:

VYEADDVILHT (SEQ ID NO 85) VYETEHHILHL (SEQ ID NO 129) VYEADHHIMHL (SEQID NO 130) VYETDHHILHL (SEQ ID NO 131) VYEADNLILHA (SEQ ID NO 86)VWQLRAIVLHV (SEQ ID NO 232) VYEADYHILHL (SEQ ID NO 233) VYETDNHILHL (SEQID NO 234) VYETENHILHL (SEQ ID NO 235) VFETVHHILHL (SEQ ID NO 236)VFETEHHILHL (SEQ ID NO 237) VFETDHHIMHL (SEQ ID NO 238) VYETENHILHL (SEQID NO 239) VYEADALILHA (SEQ ID NO 240)

Region V3 encompasses the amino acids 230 to 242. The following uniqueV3 region sequences can be deduced from FIG. 5:

VQDGNTSTCWTPV (SEQ ID NO 87) VQDGNTSACWTPV (SEQ ID NO 241) VRVGNQSRCWVAL(SEQ ID NO 132) VRTGNTSRCWVPL (SEQ ID NO 133) VRAGNVSRCWTPV (SEQ ID NO134) EEKGNISRCWIPV (SEQ ID NO 242) VKTGNQSRCWVAL (SEQ ID NO 243)VRTGNQSRCWVAL (SEQ ID NO 244) VKTGNQSRCWIAL (SEQ ID NO 245)VKTGNVSRCWIPL (SEQ ID NO 247) VKTGNVSRCWISL (SEQ ID NO 248)VRKDNVSRCWVQI (SEQ ID NO 249)

Region V4 encompasses the amino acids 248 to 257. The following uniqueV4 region sequences can be deduced from FIG. 5:

VRYVGATTAS (SEQ ID NO 89) APYIGAPLES (SEQ ID NO 135) APYVGAPLES (SEQ IDNO 136) AVSMDAPLES (SEQ ID NO 137) APSLGAVTAP (SEQ ID NO 90) APSFGAVTAP(SEQ ID NO 250) VSQPGALTKG (SEQ ID NO 251) VKYVGATTAS (SEQ ID NO 252)APYIGAPVES (SEQ ID NO 253) AQHLNAPLES (SEQ ID NO 254) SPYVGAPLEP (SEQ IDNO 255) SPYAGAPLEP (SEQ ID NO 256) APYLGAPLEP (SEQ ID NO 257) APYLGAPLES(SEQ ID NO 258) APYVGAPLES (SEQ ID NO 259) VPYLGAPLTS (SEQ ID NO 260)APHLRAPLSS (SEQ ID NO 261) APYLGAPLTS (SEQ ID NO 262)

Region V5 encompasses the amino acids 294 to 303. The following uniqueV5 region peptides can be deduced from FIG. 5:

RPRRHQTVQT (SEQ ID NO 91) QPRRHWTTQD (SEQ ID NO 138) RPRRHWTTQD (SEQ IDNO 139) RPRQHATVQN (SEQ ID NO 92) RPRQHATVQD (SEQ ID NO 263) SPQHHKFVQD(SEQ ID NO 264) RPRRLWTTQE (SEQ ID NO 265) PPRIHETTQD (SEQ ID NO 266)

The variable region in the E2 region (HVR-2) of type 5a as shown in FIG.12 spanning amino acid positions 471 to 484 is also a preferred peptideaccording to the present invention with the following sequence:

TISYANGSGPSDDK (SEQ ID NO 267)

The above given list of peptides are particularly suitable for vaccineand diagnostic development.

Also comprised in the present invention is any synthetic peptide orpolypeptide containing at least 5 contiguous amino acids derived fromthe above-defined peptides in their peptidic chain.

According to a specific embodiment, the present invention relates to acomposition as defined above, wherein said contiguous sequence isselected from any of the following HCV amino acid type 3 sequences:

-   -   a sequence having a homology of more than 72%, preferably more        than 74%, more preferably more than 77% and most preferably more        than 80 or 84% homology to any of the amino acid sequences as        represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26 or 28 (HD10,        BR36, BR33 sequences) in the region spanning positions 140 to        319 in the Core/E1 region as shown in FIG. 5;    -   a sequence having a homology of more than 70%, preferably more        than 72%, more preferably more than 75% homology, most        preferably more than 81% homology to any of the amino acid        sequences as represented in SEQ ID NO 14, 16, 18, 20, 22, 24, 26        or 28 (HD10, BR36, BR33 sequences) in the E1 region spanning        positions 192 to 319 as shown in FIG. 5;    -   a sequence having a homology of more than 86%, preferably more        than 88%, and most preferably more than 90% homology to the        amino acid sequences as represented in SEQ ID NO 148 (type 3c);        BE98 in the region spanning positions 1 to 110 in the Core        region as shown in FIG. 5;    -   a sequence having a homology of more than 76%, preferably more        than 78%, most preferably more than 80% to any of the amino acid        sequences as represented in SEQ ID NO 30, 32, 34, 36, 38 or 40        (HCC153, HD10, BR36 sequences) in the region spanning positions        1646 to 1764 in the NS3/NS4 region as shown in FIGS. 7 and 11;    -   a sequence having a homology of more than 81%, preferably more        than 83%, and most preferably more than 86% homology to any of        the amino acid sequences as represented in SEQ ID NO 14, 16, 18,        20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the region        spanning positions 140 to 319 in the Core/E1 region as shown in        FIG. 5;    -   a sequence having a homology of more than 81.5%, preferably more        than 83%, and most preferably more than 86% homology to any of        the amino acid sequences as represented in SEQ ID NO 14, 16, 18,        20, 22, 24, 26 or 28 (HD10, BR36, BR33 sequences) in the E1        region spanning positions 192 to 319 as shown in FIG. 5;    -   a sequence having a homology of more than 86%, preferably more        than 88%, most preferably more than 90% to the amino acid        sequence as represented in SEQ ID NO 150; (type 3c BE98) in the        region spanning positions 2645 to 2757 in the NS5B region as        shown in FIG. 2.

According to yet another embodiment, the present invention relates to acomposition as defined above, wherein said contiguous sequence isselected from any of the following HCV amino acid type 4 sequences:

-   -   a sequence having a homology of more than 80%, preferably more        than 82%, most preferably more than 84% homology to any of the        amino acid sequences as represented in SEQ ID NO 119, 121, and        123 (GB358, GB549, GB809 sequences) in the region spanning        positions 127 to 319 of the Core/E1 region as shown in FIG. 5;    -   a sequence having a homology of more than 73%, preferably more        than 75%, most preferably more than 78% homology in the E1        region spanning positions 192 to 319 to any of the amino acid        sequences as represented in SEQ ID NO 119, 121, and 119, 121,        and 123 (GB358, GB549, GB809 sequences) in the region spanning        positions 140 to 319 of the Core/E1 region as shown in FIG. 5;    -   a sequence having more than 85%, preferably more than 86%, most        preferably more than 87% homology to any of the amino acid        sequences as represented in SEQ ID NO 119, 121 or 123 (GB358,        GB549, GB809 sequences) in the region spanning positions 192 to        319 of E1 as shown in FIG. 5;    -   a sequence showing more than 73%, preferably more than 74%, most        preferably more than 75% homology to any of the amino acid        sequences as represented in SEQ ID NO 107, 109, 111, 113, 115 or        117 (GB48, GB116, GB215, GB358, GB549, GB809 sequences) in the        region spanning positions 2645 to 2757 of the NS5B region as        shown in FIG. 2;    -   a sequence having any of the sequences as represented in SEQ ID        NO 164 or 166 (GB809 and CAM600 sequences) in the Core region as        shown in FIG. 5;    -   a sequence having any of the sequences as represented in SEQ ID        NO 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 or 190        (CAM600, GB809, CAMG22, CAMG27, GB549, GB438, CAR4/1205,        CAR4/901, GB116, GB215, GB958, GB809-4 sequences) in the Core/E1        region as shown in FIG. 5;    -   a sequence having any of the sequences as represented in SEQ ID        NO 194, 196, 202, 204, 206, 208, 210, 212 (GB724, BE100, PC,        CAM600, CAMG22, etc.) in the NS5B region or in SEQ ID NOs: 198,        200 in the NS3/4 region.

The above-mentioned type 4 peptides polypeptides comprise at least anamino acid sequence selected from any HCV type 4 polyprotein with theexception of core sequence as disclosed by Simmonds et al. (1993, EG-29,see FIG. 5).

According to yet another aspect, the present invention relates to acomposition as defined above, wherein said contiguous sequence isselected from any of the following HCV amino acid type 5 sequences:

-   -   a sequence having more than 93%, preferably more than 94%, most        preferably more than 95% homology in the region spanning Core        positions 1 to 191 to any of the amino acid sequences as        represented in SEQ ID NO 42, 44, 46, 48, 50, 52 or 54 (PC        sequences) and SEQ ID NO 152 (BE95) as shown in FIG. 5;    -   a sequence having more than 73%, preferably more than 74%, most        preferably more than 76% homology in the region spanning E1        positions 192 to 319 to any of the amino acid sequences as        represented in SEQ ID NO 42, 44, 46, 48, 50, 52 or 54 (PC        sequences) as shown in FIG. 5;    -   a sequence having a more than 78%, preferably more than 80%,        most preferably more than 83% homology to any of the amino acid        sequences as represented in SEQ ID NO 42, 44, 46, 48, 50, 52,        54, 154, 156 (BE95, BE100) (PC sequences) in the region spanning        positions 1 to 319 of the Core/E1 region as shown in FIG. 5;    -   a sequence having more than 90%, preferably more than 91%, most        preferably more than 92% homology to any of the amino acid        sequences represented in SEQ ID NO 56 or 58 (PC sequences) in        the region spanning positions 1286 to 1403 of the NS3 region as        shown in FIG. 7 or 11;    -   a sequence having more than 66%, more particularly 68%, most        particularly 70% or more homology to any of the amino acid        sequences as represented in SEQ ID NO 60 or 62 (PC sequences) in        the region spanning positions 1646 to 1764 of the NS3/4 region        as shown in FIG. 7 or 11.

According to yet another embodiment, the present invention relates to acomposition as defined above, wherein said contiguous sequence isselected from any of the following HCV amino acid type 2d sequences:

-   -   a sequence having more than 83%, preferably more than 85%, most        preferably more than 87% homology to the amino acid sequence as        represented in SEQ ID NO 144 (NE92) in the region spanning        positions 1 to 319 of the Core/E1 region as shown in FIG. 5;    -   a sequence having more than 79%, preferably more than 81%, most        preferably more than 84% homology in the region spanning E1        positions 192 to 319 to the amino acid sequence as represented        in SEQ ID NO 144 (NE92) as shown in FIG. 12;    -   a sequence having more than 95%, more particularly 96%, most        particularly 97% or more homology to the amino acid sequence as        represented in SEQ ID NO 146 (NE92) in the region spanning        positions 2645 to 2757 of the NS5B region as shown in FIG. 2.

The present invention also relates to a recombinant vector, particularlyfor cloning and/or expression, with said recombinant vector comprising avector sequence, an appropriate prokaryotic, eukaryotic or viralpromoter sequence followed by the nucleotide sequences as defined above,with said recombinant vector allowing the expression of any one of theHCV type 2 and/or HCV type 3 and/or type 4 and/or type 5 derivedpolypeptides as defined above in a prokaryotic, or eukaryotic host or inliving mammals when injected as naked DNA, and more particularly arecombinant vector allowing the expression of any of the following HCVtype 2d, type 3, type 4 or type 5 polypeptides spanning the followingamino acid positions:

-   -   a polypeptide starting at position 1 and ending at any position        in the region between positions 70 and 326, more particularly a        polypeptide spanning positions 1 to 70, 1 to 85, positions 1 to        120, positions 1 to 150, positions 1 to 191, positions 1 to 200,        for expression of the Core protein, and a polypeptide spanning        positions 1 to 263, positions 1 to 326, for expression of the        Core and E1 protein;    -   a polypeptide starting at any position in the region between        positions 117 and 192, and ending at any position in the region        between positions 263 and 326, for expression of E1, or forms        that have the putative membrane anchor deleted (positions 264 to        293 plus or minus 8 amino acids);    -   a polypeptide starting at any position in the region between        positions 1556 and 1688, and ending at any position in the        region between positions 1739 and 1764, for expression of the        NS4 regions, more particularly a polypeptide starting at        position 1658 and ending at position 1711 for expression of the        NS4a antigen, and more particularly, a polypeptide starting at        position 1712 and ending between positions 1743 and 1972, for        example 1712–1743, 1712–1764, 1712–1782, 1712–1972, 1712 to 1782        and 1902 to 1972 for expression of the NS4b protein or parts        thereof.

The term “vector” may comprise a plasmid, a cosmid, a phage, or a virus.

In order to carry out the expression of the polypeptides of theinvention in bacteria such as E. coli or in eukaryotic cells such as inS. cerevisiae, or in cultured vertebrate or invertebrate hosts such asinsect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, thefollowing steps are carried out:

-   -   transformation of an appropriate cellular host with a        recombinant vector, in which a nucleotide sequence coding for        one of the polypeptides of the invention has been inserted under        the control of the appropriate regulatory elements, particularly        a promoter recognized by the polymerases of the cellular host        and, in the case of a prokaryotic host, an appropriate ribosome        binding site (RBS), enabling the expression in said cellular        host of said nucleotide sequence. In the case of an eukaryotic        host any artificial signal sequence or pre/pro sequence might be        provided, or the natural HCV signal sequence might be employed,        e.g. for expression of E1 the signal sequence starting between        amino acid positions 117 and 170 and ending at amino acid        position 191 can be used, for expression of NS4, the signal        sequence starting between amino acid positions 1646 and 1659 can        be used, culture of said transformed cellular host under        conditions enabling the expression of said insert.

The present invention also relates to a composition as defined above,wherein said polypeptide is a recombinant polypeptide expressed by meansof an expression vector as defined above.

The present invention also relates to a composition as defined above,for use in a method for immunizing a mammal, preferably humans, againstHCV comprising administring a sufficient amount of the compositionpossibly accompanied by pharmaceutically acceptable adjuvants, toproduce an immune response, more particularly a vaccine compositionincluding HCV type 3 polypeptides derived from the Core, E1 or the NS4region and/or HCV type 4 and/or HCV type 5 polypeptides and/or HCV type2d polypeptides.

The present invention also relates to an antibody raised uponimmunization with a composition as defined above by means of a processas defined above, with said antibody being reactive with any of thepolypeptides as defined above, and with said antibody being preferably amonoclonal antibody.

The monoclonal antibodies of the invention can be produced by anyhybridoma liable to be formed according to classical methods fromsplenic cells of an aniunal, particularly from a mouse or rat, immunizedagainst the HCV polypeptides according to the invention, or muteinsthereof, or fragments thereof as defined above on the one hand, and ofcells of a myeloma cell line on the other hand, and to be selected bythe ability of the hybridoma to produce the monoclonal antibodiesrecognizing the polypeptides which has been initially used for theimmunization of the animals.

The antibodies involved in the invention can be labelled by anappropriate label of the enzymatic, fluorescent, or radioactive type.

The monoclonal antibodies according to this preferred embodiment of theinvention may be humanized versions of mouse monoclonal antibodies madeby means of recombinant DNA technology, departing from parts of mouseand/or human genomic DNA sequences coding for H and L chains or fromcDNA clones coding for H and L chains.

Alternatively the monoclonal antibodies according to this preferredembodiment of the invention may be human monoclonal antibodies. Theseantibodies according to the present embodiment of the invention can alsobe derived from human peripheral blood lymphocytes of patients infectedwith type 3, type 4 or type 5 HCV, or vaccinated against HCV. Such humanmonoclonal antibodies are prepared, for instance, by means of humanperipheral blood lymphocytes (PBL) repopulation of severe combinedimmune deficiency (SCID) mice (for recent review, see Duchosal et al.1992).

The invention also relates to the use of the proteins of the invention,muteins thereof, or peptides derived therefrom for the selection ofrecombinant antibodies by the process of repertoire cloning (Persson etal., 1991).

Antibodies directed to peptides derived from a certaing genotype may beused either for the detection of such HCV genotypes, or as therapeuticagents.

The present invention also relates to the use of a composition asdefined above for incorporation into an immunoassay for detecting HCV,present in biological sample liable to contain it, comprising at leastthe following steps:

-   -   (i) contacting the biological sample to be analyzed for the        presence of HCV antibodies with any of the compositions as        defined above preferably in an immobilized form under        appropriate conditions which allow the formation of an immune        complex, wherein said polypeptide can be a biotinylated        polypeptide which is covalently bound to a solid substrate by        means of streptavidin or avidin complexes,    -   (ii) removing unbound components,    -   (iii) incubating the immune complexes formed with heterologous        antibodies, which specifically bind to the antibodies present in        the sample to be analyzed, with said heterologous antibodies        having conjugated to a detectable label under appropriate        conditions,    -   (iv) detecting the presence of said immunecomplexes visually or        by means of densitometry and inferring the HCV serotype present        from the observed hybridization pattern.

The present invention also relates to the use of a composition asdefined above, for incorporation into a serotyping assay for detectingone or more serological types of HCV present in a biological sampleliable to contain it, more particularly for detecting E1 and NS4antigens or antibodies of the different types to be detected combined inone assay format, comprising at least the following steps:

-   -   (i) contacting the biological sample to be analyzed for the        presence of HCV antibodies or antigens of one or more        serological types, with at least one of the compositions as        defined above, an immobilized form under appropriate conditions        which allow the formation of an immunecomplex,    -   (ii) removing unbound components,    -   (iii) incubating the immunecomplexes formed with heterologous        antibodies, which specifically bind to the antibodies present in        the sample to be analyzed, with said heterologous antibodies        having conjugated to a detectable label under appropriate        conditions,    -   (iv) detecting the presence of said immunecomplexes visually or        by means of densitometry and inferring the presence of one or        more HCV serological types present from the observed binding        pattern.

The present invention also relates to the use of a composition asdefined above, for immobilization on a solid substrate and incorporationinto a reversed phase hybridization assay, preferably for immobilizationas parallel lines onto a solid support such as a membrane strip, fordetermining the presence or the genotype of HCV according to a method asdefined above.

The present invention thus also relates to a kit for determining thepresence of HCV genotypes as defined above present in a biologicalsample liable to contain them, comprising:

-   -   possibly at least one primer composition containing any primer        selected from those defined above or any other HCV type 3 and/or        HCV type 4, and/or HCV type 5, or universal HCV primers,    -   at least one probe composition as defined above, with said        probes being preferentially immobilized on a solid substrate,        and more preferentially on one and the same membrane strip,    -   a buffer or components necessary for producing the buffer        enabling hybridization reaction between these probes and the        possibly amplified products to be carried out,    -   means for detecting the hybrids resulting from the preceding        hybriziation,    -   possibly also including an automated scanning and interpretation        device for inferring the HCV genotypes present in the sample        from the observed hybridization pattern.

The genotype may also be detected by means of a type-specific antibodyas defined above, which is linked to any polynucleotide sequence thatcan afterwards be amplified by PCR to detect the immune complex formed(Immuno-PCR, Sano et al., 1992);

The present invention also relates to a kit for determining the presenceof HCV antibodies as defined above present in a biological sample liableto contain them, comprising:

-   -   at least one polypeptide composition as defined above,        preferentially in combination with other polypeptides or        peptides from HCV type 1, HCV type 2 or other types of HCV, with        said polypeptides being preferentially immobilized on a solid        substrate, and more preferentially on one and the same membrane        strip,    -   a buffer or components necessary for producing the buffer        enabling binding reaction between these polypeptides and the        antibodies against HCV present in the biological sample,    -   means for detecting the immunecomplexes formed in the preceding        binding reaction,    -   possibly also including an automated scanning and interpretation        device for inferring the HCV genotypes present in the sample        from the observed binding pattern.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Alignment of consensus nucleotide sequences for each of the type 3aisolates BR34, BR36, and BR33, deduced from the clones with SEQ ID NO 1,5, 9; type 4 isolates GB48, GB116, GB215, GB358, GB549, GB809, CAM600,CAMG22, GB438, CAR4/1205, CAR1/501 (SEQ ID NO. 106, 108, 110, 112, 114,116, 201, 203, 205, 207, 209 and 211); type 5a isolates BE95 and BE96(SEQ ID NO 159 and 161) and type 2d isolate NE92 (SEQ ID NO 145) fromthe region between nucleotides 7932 and 8271, with known sequences fromthe corresponding region of isolates HCV-1, HCV-J, HC-J6, HC-J8, T1 andT9, and others as shown in Table 3.

FIG. 2

Alignment of amino acids sequences deduced from the nucleic acidsequences as represented in FIG. 1 from the subtype 3a clones BR34 (SEQID NO 2, 4), BR36 (SEQ ID NO 6, 8) and BR33 (SEQ ID NO 10, 12), thesubtype 3c clone BE98 (SEQ ID NO 150), and the type 4 clones GB48 (SEQID NO 107), GB116 (SEQ ID NO 109), GB215 (SEQ ID NO 111), GB358 (SEQ IDNO 113), GB549 (SEQ ID NO 115) GB809 (SEQ ID NO 117); CAM600, CAMG22,GB438, CAR4/1205, CAR1/501 (SEQ ID NO 202, 204, 206, 208, 210, 212); thetype 5a clones BE95 and BE96 (SEQ ID NO 160 and 162); as well as thesubtype 2d isolate NE92 (SEQ ID NO 146) from the region between aminoacids 2645 to 2757 with known sequences from the corresponding region ofisolates HCV-I, HCV-J, HC-J6, and HC-J8, T1 and T9, and other sequencesas shown in Table 3.

FIG. 3

Aligment of type 2d, 3c, 4 and 5a nucleotide sequences from isolatesNE92, BE98, GB358, GB809, CAM600, GB724, BE95 (SEQ ID NO 143, 147, 191,163, 165, 193 and 151) in the Core region between nucleotide positions 1and 500, with known sequences from the corresponding region of type 1,type 2, type 3 and type 4 sequences.

FIG. 4

Alignment of nucleotide sequences for the subtype 2d isolate NE92 (SEQID NO 143), the type 4 isolates GB358 (SEQ ID NO 118 and 187), GB549(SEQ ID NO 120 and 175), and GB809-2 (SEQ ID NO 122 and 169), GB 809-4,BG116, GB215, CAM600, CAMG22, CAMG27, GB438, CAR4/1205, CAR4/901 (SEQ IDNO 189, 183, 185, 167, 171, 173, 177, 179, 181), sequences for each ofthe subtype 3a isolates HD10, BR36, and BR33, (SEQ ID NO 13, 15, 17(HD10), 19, 21 (BR36) and 23, 25 or 27 (BR23) and the subtype 5aisolates BE95 and BE100 (SEQ ID NO 143 and 195) from the region betweennucleotides 379 and 957, with known sequences from the correspondingregion of type 1 and 2 and 3.

FIG. 5

Alignment of amino acid sequences deduced from the new HCV nucleotidesequences of the Core/E1 region of isolates BR33, BR36, HD10, GB358,GB549, and GB809, PC or BE95, CAM600, and GB724 (SEQ ID NO. 14, 20, 24,119 or 192, 121, 123 or 164, 54 or 152, 166 and 194) from the regionbetween positions 1 and 319, with known sequences from type 1a (HCV-1),type 1b (HCV-J), type 2a (HC-JG), type 2b (HC-J8), NZL1, HCV-TR,positions 7–89 of type 3a (E-b1), and positions 8–88 of type 4a (EG-29).V-Core, variable region with type-specific features in the core protein,V1, variable region 1 of the E1 protein, V2, variable region 2 of the E1protein, V3, variable region 3 of the E1 protein, V4, variable region 4of the E1 protein, V5, variable region 5 of the E1 protein.

FIG. 6

Alignment of nucleotide sequences of isolates HCCL53, HD10 and BR36,deduced from clones with SEQ ID NO 29, 31, 33, 35, 37 and 39, from theNS3/4 region between nucleotides 4664 to 5292, with known sequences fromthe corresponding region of isolates HCV-1, HCV-J, HC-J6, and HC-J8,EB1, EB2, EB6 and EB7.

FIG. 7

Alignment of amino acid sequences deduced from the new HCV nucleotidesequences of the NS3/NS4 region of isolate BR36 (SEQ ID NO 36, 38 and40) and BE95 (SEQ ID NO 270). NS4-1, indicates the region that wassynthesized as synthetic peptide 1 of the NS4 region, NS4-5, indicatesthe region that was synthesized as synthetic peptide 5 of the NS4region; NS4-7, indicates the region that was synthesized as syntheticpeptide 7 of the NS4 region.

FIG. 8

Reactivity of the three LIPA-selected (Stuyver et al., 1993) type 3 seraon the Inno-LIA HCV Ab II assay (Innogenetics) (left), and on theNS4-LIA test. For the NS4-LIA test, NS4-1, NS4-5, and NS4-7 peptideswere synthesized based on the type 1 (HCV-1), type 2 (HC-J6) and type 3(BR36) prototype isolate sequences as shown in Table 4, and applied asparallel lines onto a membrane strip as indicated. 1, serum BR33, 2,serum HD10, 3, serum DKH.

FIG. 9

Nucleotide sequences of Core/E1 clones obtained from the PCR fragmentsPC-2, PC-3, and PC-4, obtained from serum BE95 (PC-2-1 (SEQ ID NO 41),PC-2-6 (SEQ ID NO 43), PC-4-1 (SEQ ID NO 45), PC-4-6 (SEQ ID NO 47),PC-34 (SEQ ID NO 49), and PC-3-8 (SEQ ID NO 51)) of subtype 5a isolateBE95.

A consensus sequence is shown for the Core and E1 region of isolateBE95, presented as PC C/E1 with SEQ ID NO 53. Y, C or T, R, A or G, S, Cor G.

FIG. 10

Alignment of nucleotide sequences of clones with SEQ ID NO 197 and 199(PC sequences, see also SEQ ID NO 55, 57, 59) and SEQ ID NO 35, 37 and39 (BR36 sequences) from the NS3/4 region between nucleotides 3856 to5292, with known sequences from the corresponding region of isolatesHCV-1, HCV-J, HC-J6, and HC-J8.

FIG. 11

Alignment of amino acid sequences of subtype 5a BE95 isolate PC cloneswith SEQ ID NO 56 and 58, from the NS3/4 region between amino acids 1286to 1764, with known sequences from the corresponding region of isolatesHCV-1, HCV-J, HC-J6, and HC-J8.

FIG. 12

Aligment of amino acid sequences of subtype 5a isolate BE95 (SEQ ID NO158) in the E1/E2 region spanning positions 328 to 546, with knownsequnces from the corresponding region of isolates HCV-1, HCV-J, HC-J6,HC-J8, NZL1 and HCV-TR (see Table 3).

FIG. 13

Alignment of the nucleotide sequences of subtype 5a isolate BE95 (SEQ IDNO 157) in the E1/E2 region with known HCV sequences as shown in Table3.

EXAMPLES Example 1 The NS5b Region of HCV Type 3

Type 3 sera, selected by means of the INNO-LiPA HCV research kit(Stuyver et al., 1993) from a number of Brazilian blood donors, werepositive in the HCV antibody ELISA (Innotest HCV Ab II; Innogenetics)and/or in the INNO-LIA HCV Ab II confirmation test (Innogenetics). Onlythose sera that were positive after the first round of PCR reactions(Stuyver et al., 1993) were retained for further study.

Reverse transcription and nested PCR: RNA was extracted from 50 μl serumand subjected to cDNA synthesis as described (Stuyver et al., 1993).This cDNA was used as template for PCR, for which the total volume wasincreased to 50 μl containing 10 pmoles of each primer, 3 μl of 10×Pfubuffer 2 (Stratagene) and 2.5 U of Pfu DNA polymerase (Stratagene). ThecDNA was amplified over 45 cycles consisting of 1 min 94° C., 1 min 50°C. and 2 min 72° C. The amplified products were separated byelectrophoresis, isolated, cloned and sequenced as described (Stuyver etal., 1993).

Type 3a and 3b-specific primers in the NS5 region were selected from thepublished sequences (Mori et al., 1992) as follows:

-   -   for type 3a:

HCPr161(+): 5′-ACCGGAGGCCAGGAGAGTGATCTCCTCC-3′ (SEQ ID NO 63) andHCPr162(−): 5′-GGGCTGCTCTATCCTCATCGACGCCATC-3′ (SEQ ID NO 64);

-   -   for type 3b:

HCPr163(+): 5′-GCCAGAGGCTCGGAAGGCGATCAGCGCT-3′ (SEQ ID NO 65) andHCPr164(−): 5′-GAGCTGCTCTGTCCTCCTCGACGCCGCA-3′ (SEQ ID NO 66)

Using the Line Probe Assay (LiPA) (Stuyver et al., 1993), sevenhigh-titer type 3 sera were selected and subsequently analyzed with theprimer sets HCPr161/162 for type 3a, and HCPr163/164 for type 3b. Noneof these sera was positive with the type 3b primers. NS5 PCR fragmentsobtained using the type 3a primers from serum BR36 (BR36-23), serum BR33(BR33-2) and serum BR34 (BR34-4) were selected for cloning. Thefollowing sequences were obtained from the PCR fragments:

-   -   From fragment BR34-4:

BR34-4-20 (SEQ ID NO 1), BR34-4-19 (SEQ ID NO 3)

-   -   From fragment BR36-23:

BR36-23-18 (SEQ ID NO 5), BR36-23-20 (SEQ ID NO 7)

-   -   From fragment BR33-2:

BR33-2-17 (SEQ ID NO 9), BR33-2-21 (SEQ ID NO 11)

An alignment of sequences with SEQ ID NO 1, 5 and 9 with known sequencesis given in FIG. 1. An alignment of the deduced amino acid sequences isshown in FIG. 2. The 3 isolates are very closely related to each other(mutual homologies of about 95%) and to the published sequences of type3a (Mori et al., 1992), but are only distantly related to type 1 andtype 2 sequences (Table 5). Therefore, it is clearly demonstrated thatNS5 sequences from LiPA-selected type 3 sera are indeed derived from atype 3 genome. Moreover, by analyzing the NS5 region of serum BR34, forwhich no 5′UR sequences were determined as described in Stuyver et al.(1993), the excellent correlation between typing by means of the LiPAand genotyping as deduced from nucleotide sequencing was further proven.

Example 2 The Core/E1 Region of HCV Type 3

After aligning the sequences of HCV-1 (Choo et al., 1991), HCV-J (Katoet al., 1990), HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al.,1992), PCR primers were chosen in those regions of little sequencevariation. Primers HCPr23(+): 5′-CTCATGGGGTACATTCCGCT-3′ (SEQ ID NO 67)and HCPr54(−): 5′-TATTACCAGTTCATCATCATATCCCA-3′ (SEQ ID NO 68), weresynthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). This setof primers was selected to amplify the sequence from nucleotide 397 to957 encoding amino acids 140 to 319 (Kato et al., 1990): 52 amino acidsfrom the carboxyterminus of core and 128 amino acids of E1 (Kato et al.,1990). The amplification products BR36-9, BRR33-1, and HD10-2 werecloned as described (Stuyver et al., 1993). The following clones wereobtained from the PCR fragments:

-   -   From fragment HD10-2:

HD10-2-5 (SEQ ID NO 13), HD10-2-14 (SEQ ID NO 15), HD10-2-21 (SEQ ID NO17)

-   -   From fragment BR36-9:

BR36-9-13 (SEQ ID NO 19), BR36-9-20 (SEQ ID NO 21),

-   -   From fragment BR33-1:

BR33-1-10 (SEQ ID NO 23), BR33-1-19 (SEQ ID NO 25), BR33-1-20 (SEQ ID NO27),

An alignment of the type 3 E1 nucleotide sequences (HD10, BR36, BR33)with SEQ ID NO 13, 19 and 23 with known E1 sequences is presented inFIG. 4. Four variations were detected in the E1 clones from serum HD10and BR36, while only 2 were found in BR33. All are silent third lettervariations, with the exception of mutations at position 40 (L to P) and125 (M to I). The homologies of the type 3 E1 region (without core) withtype 1 and 2 prototype sequences are depicted in Table 5.

In total, 8 clones covering the core/E1 region of 3 different isolateswere sequenced and the E1 portion was compared with the known genotypes(Table 3) as shown in FIG. 5. After computer analysis of the deducedamino acid sequence, a signal-anchor sequence at the corecarboxyterminus was detected which might, through analogy with type 1b(Hijikata et al., 1991), promote cleavage before the LEWRN sequence(position 192, FIG. 5; SEQ ID NO:271). The L-to-P mutation in one of theHD10-2 clones resides in this signal-anchor region and potentiallyimpairs recognition by signal peptidase (computer prediction). Since noexamples of such substitutions were found at this position in previouslydescribed sequences, this mutation might have resulted from reversetranscriptase or Pu polymerase misincorporation. The 4 amino-terminalpotential N-linked glycosylation sites, which are also present in HCVtypes 1a and 2, remain conserved in type 3. The N-glycosylation site intype 1b (aa 250, Kato et al., 1990) remains a unique feature of thissubtype. All E1 cysteines, and the putative transmembrane region (aa 264to 293, computer prediction) containing the aspartic acid at position279, are conserved in all three HCV types. The following hypervariableregions can be delineated: V1 from aa 192 to 203 (numbering according toKato et al., 1990), V2 (213–223), V3 (230–242), V4 (248–257), and V5(294–303). Such hydrophilic regions are thought to be exposed to thehost defense mechanisms. This variability might therefore have beeninduced by the host's immune response. Additional putative N-linkedglycosylation sites in the V4 region in all type 1b isolates known todayand in the V5 region of HC-J8 (type 2b) possibly further contribute tomodulation of the immune response. Therefore, analysis of this region,in the present invention, for type 3 and 4 sequences has beeninstrumental in the delineation of epitopes that reside in the V-regionsof E1, which will be critical for future vaccine and diagnosticsdevelopment.

Example 3 The NS3/NS4 Region of HCV Type 3

For the NS3/NS4 border region, the folllowing sets of primers wereselected in the regions of little sequence variability after aligningthe sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990),HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992) (smallercase lettering is used for nucleotides added for cloning purposes):

-   -   set A:

HCPr116(+): 5′-ttttAAATACATCATGRCITGYATG-3′ (SEQ ID NO 69)

HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) setB: HCPr116(+): 5′-ttttAAATACATCATGRCITGYATG-3′ (SEQ ID NO 69)HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71)set C: HCPr117(+): 5′-ttttAAATACATCGCIRCITGCATGCA-3′ (SEQ ID NO 72)HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) setD: HCPr117(+): 5′-ttttAAATACATCGCIRCITGCATGCA-3′ (SEQ ID NO 72)HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71)set E: HCPr116(+): 5′-ttttAAATACATCATGRCITGYATG-3′ (SEQ ID NO 69)HCPr119(−): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3′ (SEQ ID NO 73)set F: HCPr117(+): 5′-ttttAAATACATCGCIRCITGCATGCA-3′ (SEQ ID NO 72)HCPr119(−): actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3′ (SEQ ID NO 73)set G: HCPr131(+): 5′-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3′ (SEQ IDNO 74) HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO70) set H: HCPr130(+): 5′-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3′ (SEQID NO 75) HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO70) set I: HCPr134(+): 5′-CATATAGATGCCCACTTCCTATC-3′ (SEQ ID NO 76)HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) setJ: HCPr131(+): 5′-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3′ (SEQ ID NO74) HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO71) set K: HCPr130(+): 5′-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3′ (SEQID NO 75) HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQID NO 71) set L: HCPr134(+): 5′-CATATAGATGCCCACTTCCTATC-3′ (SEQ ID NO76) HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO71) set M: HCPr3(+): 5′-GTGTGCCAGGACCATC-3′ (SEQ ID NO 77) and HCPr4(−):5′-GACATGCATGTCATGATGTA-3 (SEQ ID NO 78) set N: HCPr3(+):5′-GTGTGCCAGGACCATC-3′ (SEQ ID NO 77) and HCPr118(−):5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71) set O:HCPr3(+): 5′-GTGTGCCAGGACCATC-3′ (SEQ ID NO 77) and HCPr66 (−):5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70)

No PCR products could be obtained with the sets of primers A, B, C, D,E, F, G, H, I, J, K, L, M, and N, on random-primed cDNA obtained fromtype 3 sera. With the primer set O no fragment could be amplified fromtype 3 sera. However, a smear containing a few weakly stainable bandswas obtained from serum BR36. After sequence analysis of several DNAfragments, purified and cloned from the area around 300 bp on theagarose gel, only one clone, HCC153 (SEQ ID NO 29), was shown to containHCV information. This sequence was used to design primer HCPr152.

A new primer set P was subsequently tested on several sera.

-   -   set P:

HCPr152(+): 5′-TACGCCTCTTCTATATCGGTTGGGGCCTG-3′ (SEQ ID NO 79) andHCPr66(−): 5′-CTATTATTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70)

The 464-bp HCPr152/66 fragment was obtained from serum BR36 (BR36-20)and serum HD10 (HD10-1). The following clones were obtained from thesePCR products:

-   -   From fragment HD10-1:

HD10-1-25 (SEQ ID NO 31), HD10-1-3 (SEQ ID NO 33),

-   -   From fragment BR36-20:

BR36-20-164 (SEQ ID NO 35), BR36-20-165 (SEQ ID NO 37), BR36-20-166 (SEQID NO 39),

The nucleotide sequences obtained from clones with SEQ ID NO 29, 31, 33,35, 37 or 39 are shown aligned with the sequences of prototype isolatesof other types of HCV in FIG. 6. In addition to one silent 3rd lettervariation, one 2nd letter mutation resulted in an E to G substitution atposition 175 of the deduced amino acid sequence of BR36 (FIG. 7). SerumHD10 clones were completely identical. The two type 3 isolates werenearly 94% homologous in this NS4 region. The homologies with othertypes are presented in Table 5.

Example 4 Analysis of the Anti-NS4 Response to Type-specific Peptides

As the NS4 sequence contains the information for an important epitopecluster, and since antibodies towards this region seem to exhibit littlecross-reactivity (Chan et al., 1991), it was worthwhile to investigatethe type-specific antibody response to this region. For each of the 3genotypes, HCV-1 (Choo et al., 1991), HC-J6 (Okamoto et al., 1991) andBR36 (present invention), three 20-mer peptides were synthesizedcovering the epitope region between amino acids 1688 and 1743 (asdepicted in table 6). The synthetic peptides were applied as parallellines onto membrane strips. Detection of anti-NS4 antibodies and colordevelopment was performed according to the procedure described for theINNO-LIA HCV Ab 11 kit (Innogenetics, Antwerp). Peptide synthesis wascarried out on a 9050 PepSynthesizer (Millipore). After incubation with15 LiPA-selected type 3 sera, 9 samples showed reactivity towards NS4peptides of at least 2 different types, but a clearly positive reactionwas observed for 3 sera (serum BR33, HD30 and DKH) on the type 3peptides, while negative (serum BR33 and HD30) or indeterminate (serumDKH) on the type 1and type 2 NS4 peptides; 3 sera tested negative foranti-NS4 antibodies (FIG. 8). Using the se membrane strips coated withthe 9 peptides as indicated above and as shown in FIG. 8, 38 type 1 sera(10 type 1a and 28 type 1b), 11 type 2 sera (10 type 2a and 1 type 2b),12 type 3a sera and 2 type 4 sera (as determined by the LiPA procedure)were also tested. As shown in Table 8, the sera reacted in agenotype-specific manner with the NS4 epitopes. These resultsdemonstrate that type-specific anti-NS4 antibodies can be detected inthe sera of some patients. Such genotype-specific synthetic peptidesmight be employed to develop serotyping assays, for example a mixture ofthe nine peptides as indicated above, or combined with the NS4 peptidesfrom the HCV type 4 or 6 genotype or from new genotypes corresponding tothe region between amino acids 1688 and 1743, or synthetic peptides ofthe NS4 region between amino acids 1688 and 1743 of at least one of the6 genotypes, combined with the E1 protein or deletion mutants thereof,or synthetic E1 peptides of at least one of the genotypes. Suchcompositions could be further extended with type-specific peptides orproteins, including for example the region between amino acids 68 and 91of the core protein, or more preferably the region between amino acids68 and 78. Furthermore, such type-specific antigens may beadvantageously used to improve current diagnostic screening andconfirmation assays and/or HCV vaccines.

Example 5 The Core and E1 Regions of HCV Type 5

Sample BE95 was selected from a group of sera that reacted positive in aprototype Line Probe Assay as described earlier (Stuyver et al., 1993),because a high-titer of HCV RNA could be detected, enabling cloning offragments by a single round of PCR. As no sequences from any codingregion of type 5 has been disclosed yet, synthetic oligonucleotides forPCR amplification were chosen in the regions of little sequencevariation after aligning the sequences of HCV-1 (Choo et al., 1991),HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991), HC-J8 (Okamotoet al., 1992), and the new type 3 sequences of the present inventionHD10, BR33, and BR36 (see FIG. 5, Example 2). The following sets ofprimers were synthesized on a 392 DNA/RNA synthesizer (AppliedBiosystems):

Set 1: HCPr52(+): 5′-atgTTGGGTAAGGTCATCGATACCCT-3′ (SEQ ID NO 80) andHCPr54(−): 5′-ctattaCCAGTTCATCATCATATCCCA-3′ (SEQ ID NO 78) Set 2:HCPr41(+): 5′-CCCGGGAGGTCTCGTAGACCGTGCA-3′ (SEQ ID NO 81) and HCPr40(−):5′-ctattaAAGATAGAGAAAGAGCAACCGGG-3′ (SEQ ID NO 82) Set 3: HCPr41(+):5′-CCCGGGAGGTCTCGTAGACCGTGCA-3′ (SEQ ID NO 81) and HCPr54(−):5′-ccattaCCAGTTCATCATCATATCCCA-3′ (SEQ ID NO 78)

The three sets of primers were employed to amplify the regions of thetype 5 isolate PC as described (Stuyver et al., 1993). Set 1 was used toamplify the E1 region and yielded fragment PC-4, set 2 was designed toyield the Core region and yielded fragment PC-2. Set 3 was used toamplify the Core and E1 region and yielded fragment PC-3. Thesefragments were cloned as described (Stuyver et al., 1993). The followingclones were obtained from the PCR fragments:

-   -   From fragment PC-2:

PC-2-1 (SEQ ID NO 41), PC-2-6 (SEQ ID NO 43),

-   -   From fragment PC-4:

PC-4-1 (SEQ ID NO 45), PC-4-6 (SEQ ID NO 47),

-   -   From fragment PC-3:

PC-3-4 (SEQ ID NO 49), PC-3-8 (SEQ ID NO 51)

An alignment of sequences with SEQ ID NO 41, 43, 45, 47, 49 and 51, isgiven in FIG. 9. A consensus amino acid sequence (PC C/E1; SEQ ID NO 54)can be deduced from each of the 2 clones cloned from each of the threePCR fragments as depicted in FIG. 5, which overlaps the region betweennucleotides 1 and 957 (Kato et al., 1990). The 6 clones are very closelyrelated to each other (mutual homologies of about 99.7%).

An alignment of nucleotide sequence with SEQ ID NO 53 or 151 (PC C/E1from isolate BE95) with known nucleotide sequences from the Core/E1region is given in FIG. 3. The clone is only distantly related to type1, type 2, type 3 and type 4 sequences (Table 5).

Example 6 NS3/NS4 Region of HCV Type 5

Attempts were undertaken to clone the NS3/NS4 region of the isolateBE95, described in example 5. The folllowing sets of primers wereselected in the regions of little sequence variability after aligningthe sequences of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1991),HC-J6 (Okamoto et al., 1991), and HC-J8 (Okamoto et al., 1992) and ofthe sequences obtained from type 3 sera of the present invention (SEQ IDNO 31, 33, 35, 37 and 39); smaller case lettering is used fornucleotides added for cloning purposes:

set A: HCPr116(+): 5′-ttttAAATACATCATGRCITGYATG-3′ (SEQ ID NO 66) HCPr66(−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) set B:HCPr116(+): 5′-ttttAAATACATCATGRCITGYATG-3′ (SEQ ID NO 69) HCPr118(−):5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71) set C:HCPr117(+): 5′-ttttAAATACATCGCIRCITGCATGCA-3′ (SEQ ID NO 72) HCPr66 (−):5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) set D:HCPr117(+): 5′-ttttAAATACATCGCIRCITGCATGCA-3′ (SEQ ID NO 72) HCPr118(−):5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71) set E:HCPr116(+): 5′-ttttAAATACATCATGRCITGYATG-3′ (SEQ ID NO 69) HCPr119(−):actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3′ (SEQ ID NO 73) set F:HCPr117(+): 5′-ttttAAATACATCGCIRCITGCATGCA-3′ (SEQ ID NO 72) HCPr119(−):actagtcgactaRTTIGCIATIAGCCG/TRTTCATCCAYTG-3′ (SEQ ID NO 73) set G:HCPr131(+): 5′-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3′ (SEQ ID NO 74)HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) setH: HCPr130(+): 5′-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3′ (SEQ ID NO 75)HCPr66 (−): 5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) setI: HCPr134(+): 5′-CATATAGATGCCCACTTCCTATC-3′ (SEQ ID NO 76) HCPr66 (−):5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70) set J:HCPr131(+): 5′-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3′ (SEQ ID 74)HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71)set K: HCPr130(+): 5′-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3′ (SEQ ID NO75) HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO71) set L: HCPr134(+): 5′-CATATAGATGCCCACTTCCTATC-3′ (SEQ ID NO 76)HCPr118(−): 5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71)set M: HCPr3(+): 5′-GTGTGCCAGGACCATC-3′ (SEQ ID NO 77) and HCPr4(−):5′-GACATGCATGTCATGATGTA-3′ (SEQ ID NO 78) set N: HCPr3(+):5′-GTGTGCCAGGACCATC-3′ (SEQ ID NO 77) and HCPr118(−):5′-actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ (SEQ ID NO 71) set O:HCPr3(+): 5′-GTGTGCCAGGACCATC-3′ (SEQ ID NO 77) and HCPr66 (−):5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ (SEQ ID NO 70)

No PCR products could be obtained with the sets of primers A, B, C, D,E, F, G, H, I, J, K, L, M, and N, on random-primed cDNA obtained fromtype 3 sera. However, set O yielded what appeared to be a PCR artifactfragment estimated about 1450 base pairs, instead of the expected 628base pairs. Although it is not expected that PCR artifact fragmentscontain information of the gene or genome that was targetted in theexperiment, efforts were put in cloning of this artifact fragment, whichwas designated fragment PC-1.

The following clones, were obtained from fragment PC-1:

PC-1-37 (SEQ ID NO 59 and SEQ ID NO 55), PC-1-48 (SEQ ID NO 61 and SEQID NO 57)

The sequences obtained from the 5′ and 3′ ends of the clones are givenin SEQ ID NOS 55, 57, 59, and 61, and the complete sequences with SEQ IDNO 197 and 199 are shown aligned with the sequences of prototypeisolates of other types of HCV in FIG. 10 and the alignment of thededuced amino acid sequences is shown in FIGS. 11 and 7. Surprisingly,the PCR artifact clone contained HCV information. The positions of thesequences within the HCV genome are compatible with a contiguous HCVsequence of 1437 nucleotides, which was the estimated size of the clonedPCR artifact fragment. Primer HCPr66 primed correctly at the expectedposition in the HCV genome. Therefore, primer HCPr3 must haveincidentally misprimed at a position 809 nucleotides upstream of itslegitimate position in the HCV genome. This could not be expected sinceno sequence information was available from a coding region of type 5.

Example 7 The E2 Region of HCV Type 5

Serum BE95 was chosen for experiments aimed at amplifying a part of theE2 region of HCV type 5.

After aligning the sequences of HCV-1 (2), HCV-J (1), HC-J6 (3), andHC-J8 (4), PCR primers were chosen in those regions of little sequencevariation.

Primers HCPr109(+): 5′-TGGGATATGATGATGAACTGGTC-3′ (SEQ ID NO 141) andHCPr14(−): 5′-CCAGGTACAACCGAACCAATTGCC-3′ (SEQ ID NO 142) were combinedto amplify the aminoterminal region of the E2/NS1 region, and weresynthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). Withprimers HCPr109 and HCPr14, a PCR fragment of 661 bp was generated,containing 169 nucleodtides corresponding to the E1 carboxyterminus and492 bases from the region encoding the E2 aminoterminus.

An alignment of the type 5 E1/E2 sequences with seq ID NO. 158 withknown sequences is presented in FIG. 10. The deduced protein sequencewas compared with the different genotypes (FIG. 12, amino acids328–546). In the E1 region, there were no extra structural importantmotifs found. The aminoterminal part of E2 was hypervariable whencompared with the other genotypes. All 6 N-glycosylation sites and all 7cysteine residue's were conserved in this E2 region. To preservealignment, it was necessary to introduce a gap between aa 474 and 475 asfor type 3a, but not between aa 480 and 481, as for type 2.

Example 8 The NS5b Region of HCV Type 4

Type 4 sera GB48, GB116, GB215, and GB358, selected by means of the lineprobe assay (LiPA, Stuyver et al., 1993), as well as sera GB549 andGB809 that could not be typed by means of this LiPA (only hybridizationwas observed with the universal probes), were selected from Gabonesepatients. All these sera were positive after the first round of PCRreactions for the 5′ untranslated region (Stuyver et al., 1993) and wereretained for further study.

RNA was isolated from the sera and cDNA synthesized as described inexample 1. Universal primers in the NS5 region were selected afteralignment of the published sequences as follows:

HCPr206(+): 5′-TGGGGATCCCGTATGATACCCGCTGCTTTGA-3′ (SEQ ID NO. 124) andHCPr207(−): 5′-GGCGGAATTCCTGGTCATAGCCTCCGTGAA-3′ (SEQ ID NO. 125);and were synthesized on a 392 DNA/RNA synthesizer (Applied Biosystems).Using the Line Probe Assay (LiPA), four high-titer type 4 sera and 2sera that could not be classified were selected and subsequentlyanalyzed with the primer set HCPr206/207. NS5 PCR fragments obtainedusing these primers from serum GB48 (GB48-3), serum GB116 (GB116-3),serum GB215 (GB215-3), serum GB358 (GB358-3), serum GB549 (GB549-3), andserum GB809 (GB809-3), were selected for cloning. The followingsequences were obtained from the PCR fragments:

-   From fragment GB48-3: GB48-3-10 (SEQ ID NO. 106)-   From fragment GB116-3: GB116-3-5 (SEQ ID NO. 108)-   From fragment GB215-3: GB215-3-8 (SEQ ID NO. 110)-   From fragment GB358-3: GB358-3-3 (SEQ ID NO. 112)-   From fragment GB549-3: GB549-3-6 (SEQ ID NO. 114)-   From fragment GB809-3: GB809-3-1 (SEQ ID NO. 116)

An alignment of nucleotide sequences with SEQ ID NO. 106, 108, 110, 112,114, and 116 with known sequences is given in FIG. 1. An alignment ofdeduced amino acid sequences with SEQ ID NO. 107, 109, 111, 113, 115,and 117 with known sequences is given in FIG. 2. The 4 isolates that hadbeen typed as type 4 by means of LiPA are very closely related to eachother (mutual homologies of about 95%), but are only distantly relatedto type 1, type 2, and type 3 sequences (e.g. GB358 shows homologies of65.6 to 67.7% with other genotypes, Table 4). The sequence obtained fromsera GB549 and GB809 also show similar homologies with genotypes 1, 2,and 3 (65.9 to 68.8% for GB549 and 65.0 to 68.5% for GB809, Table 4),but an intermediate homology of 79.7 to 86.8% (often observed betweensubtypes of the same type) exists between GB549 or GB809 with the groupof isolates consisting of GB48, GB116, GB215, and GB358, or betweenGB549 and GB809. These data indicate the discovery of 3 new subtypeswithin the HCV genotype 4: in the present invention, these 3 subtypesare designated subtype 4c, represented by isolates GB48, GB116, GB215,and GB358, subtype 4g, represented by isolate GB549, and subtype 4e,represented by isolate GB809. Although the homologies observed betweensubtypes in the NS5 region seem to indicate a closer relationshipbetween subtypes 4c and 4e, the homologies observed in the E1 regionindicate that subtypes 4g and 4e show the closest relation (see example8).

Example 9 The Core/E1 Region of HCV Type 4

From each of the 3 new type 4 subtypes, one representative serum wasselected for cloning experiments in the Core/E1 region. GB549 (subtype4g) and GB809 (subtype 4e) were analyzed together with isolate GB358that was chosen from the subtype 4c group.

Synthetic oligonucleotides:

After aligning the sequences of HCV-1 (2), HCV-J (1), HC-J6 (3), andHC-J8 (4), PCR primers were chosen in those regions of little sequencevariation. Primers HCPr52(+): 5′-atgTTGGGTAAGGTCATCGATACCCT-3′ (SEQ IDNO:80), HCPr23(+): 5′-CTCATGGGGTACATTCCGCT-3′ (SEQ ID NO:67), andHCPr54(−): 5′-CTATTACCAGTTCATCATCATATCCCA-3′ (SEQ ID NO:68), weresynthesized on a 392 DNA/RNA synthesizer (Applied Biosystems). The setsof primers HCPr23/54 and HCPr52/54 were used, but only with the primerset HCPr52/54, PCR fragments could be obtained. This set of primersamplified the sequence from nucleotide 379 to 957 encoding amino acids127 to 319: 65 amino acids from the carboxyterminus of core and 128amino acids of E1. The amplification products GB358-4, GB549-4, andGB809-4 were cloned as described in example 1. The following clones wereobtained from the PCR fragments:

-   From fragment GB358-4: GB358-4-1 (SEQ ID NO 118)-   From fragment GB549-4: GB549-4-3 (SEQ ID NO 120)-   From fragment GB809-4: GB809-4-3 (SEQ ID NO 122)

An alignment of the type 4 Core/E1 nucleotide sequences with seq ID NO.118, 120, and 122 with known sequences is presented in FIG. 4. Thehomologies of the type 4 E1 region (without core) with type 1, type 2,type 3, and type 5 prototype sequences are depicted in Table 4.Homologies of 53 to 66% are observed with representative isolates ofnon-type 4 genotypes. Observed homologies in the E1 region within type4, between the different subtypes, ranges from 75.2 to 78.4%. Therecently disclosed sequences of the core region of Egyptian type 4isolates (for example EG-29 in FIG. 3) described by Simmonds et al.(1993) do not allow alignment with the Gabonese sequences (as describedin the present invention) in the NSB region and may belong to differenttype 4 subtypes(s) as can be deduced from the core sequences. Thededuced amino acid sequences with SEQ ID NO 119, 121, and 123 arealigned with other prototype sequences in FIG. 5. Again, type-specificvariation mainly resides in the variable V regions, designated in thepresent invention, and therefore, type-4-specific amino acids or Vregions will be instrumental in diagnosis and therapeutics for HCV type4.

Example 10 The Core/E1and NS5b Regions of New HCV Type 2, 3 and 4Subtypes

Samples NE92 (subtype 2d), BE98 (subtype 3c), CAM600 and GB809 (subtype4e), CAMG22 and CAMG27 (subtype 4f), GB438 (subtype 4h), CAR4/1205subtype (4i), CAR1/501 (subtype 4j), CAR1/901 (subtype 4?), and GB724(subtype 4?) were selected from a group of sera that reacted positivebut aberrantly in a prototype Line Probe Assay as described earlier(Stuyver et al., 1993). Another type 5a isolate BE100 was also analyzedin the C/E1 region, and yet another type 5a isolate BE96 in the NS5bregion. A high-titer of HCV RNA could be detected, enabling cloning offragments by a single round of PCR. As no sequences from any codingregion of these subtypes had been disclosed yet, syntheticoligonucleotides for PCR amplification were chosen in the regions oflittle sequence variation after aligning the sequences of HCV-1 (Choo etal., 1991), HCV-J (Kato et al., 1990), HC-J6 (Okamoto et al., 1991),HC-J8 (Okamoto et al., 1992), and the other new sequences of the presentinvention.

The above mentioned sets 1, 2 and 3 (see example 5) of primers wereused, but only with set 1, PCR fragments could be obtained from allisolates (except for BE98, GB724, and CAR1/501). This set of primersamplified the sequence from nucleotide 379 to 957 encoding amino acids127 to 319: 65 amino acids from the carboxyterminus of core and 128amino acids of E1. With set 3, the core/E1 region from isolate NE92 andBE98 could be amplified, and with set 2, the core region of GB358,GB724, GB809, and CAM600 could be amplified. The amplification productswere cloned as described in example 1. The following clones wereobtained from the PCR fragments:

-   From isolate GB724, the clone with SEQ ID NO 193 from the core    region.-   From isolate NE92, the clone with SEQ ID NO 143-   From isolate BE98, the clone from the core/E1 region of which part    of the sequence has been analyzed and is given in SEQ ID NO 147,-   From isolate CAM600, the clone with SEQ ID NO 167 from the E1    region, or SEQ ID NO 165 from the Core/E1 region as shown in FIG. 3,-   From isolate CAMG22, the clone with SEQ ID NO 171 from the E1 region    as shown in FIG. 4,-   from isolate GB358, the clone with SEQ ID NO 191 in the core region,-   from isolate CAMG27, the clone with SEQ ID NO 173 from the core/E1    region,-   from isolate GB438, the clone with SEQ ID NO 177 from the core/E1    region,-   from isolate CAR4/1205, the clone with SEQ ID NO 179 from the    core/E1 region,-   from isolate CAR1/901, the clone with SEQ ID NO 181 from the core/E1    region,-   from isolate GB809, the clone GB809-4 with SEQ ID NO 189 from the    core/E1 region, clone GB809-2 with SEQ ID NO 169 from the core/E1    region and the clone with SEQ ID NO 163 from the core region,-   and from isolate BE100, the clone with SEQ ID NO 155 from the    Core/E1 region as shown in FIG. 4.

An alignment of these Core/E1 sequences with known Core/E1 sequences ispresented in FIG. 4. The deduced amino acid sequences with SEQ ID NO144, 148, 164, 168, 170, 172, 174, 178, 180, 182, 190, 192, 194, 156,166 are aligned with other prototype sequences in FIG. 5. Again,type-specific variation mainly resides in the variable V regions,designated in the present invention, and therefore, type 2d, 3c and type4-specific amino acids or V regions will be instrumental in diagnosisand therapeutics for HCV type (subtype) 2d, 3c or the different type 4subtypes.

The NS5b region of isolates NE92, BE98, CAM600, CAMG22, GB438,CAR4/1205, CAR1/501, and BE96 was amplified with primers HCPr206 andHCPr207 (Table 7). The corresponding clones were cloned and sequenced asin example 1 and the corresponding sequences (of which BE98 was partlysequenced) received the following identification numbers:

-   NE92: SEQ ID NO 145-   BE98: SEQ ID NO 149-   CAM600: SEQ ID NO 201-   CAMG22: SEQ ID NO 203-   GB438: SEQ ID NO 207-   CAR4/1205: SEQ ID NO 209-   CAR1/501: SEQ ID NO 211-   BE95: SEQ ID NO 159-   BE96: SEQ ID NO 161

An alignment of these NS5b sequences with known NS5b sequences ispresented in FIG. 1. The deduced amino acid sequences with SEQ ID NO146, 150, 202, 204, 206, 208, 210, 212, 160, 162 are aligned with otherprototype sequences in FIG. 2. Again, subtype-specific variations can beobserved, and therefore, type 2d, 3c and type 4-specific amino acids orV regions will be instrumental in diagnosis and therapeutics for HCVtype (subtype) 2d, 3c or the different type 4 subtypes.

Example 11 Genotype-specific Reactivity of Anti-E1Antibodies(Serotyping)

E1 proteins were expressed from vaccinia virus constructs containing acore/E1 region extending from nucleotide positions 355 to 978 (Core/E1clones described in previous examples including the primers HCPr52 andHCPr54), and expressed proteins from L119 (after the initiatormethionine) to W326 of the HCV polyprotein. The expressed protein wasmodified upon expression in the appropriate host cells (e.g. HeLa, RK13,HuTK-, HepG2) by cleavage between amino acids 191 and 192 of the HCVpolyprotein and by the addition of high-mannose type carbohydratemotifs. Therefore, a 30 to 32 kDa glycoprotein could be observed onwestern blot by means of detection with serum from patients withhepatitis C.

As a reference, a genotype 1b clone obtained form the isolate HCV-B wasalso expressed in an identical way as described above, and was expressedfrom recombinant vaccinia virus vvHCV-11A.

A panel of 104 genotyped sera was first tested for reactivity with acell lysate containing type 1b protein expressed from the recombinantvaccinia virus vvHCV-11A, and compared with cell lysate of RK13 cellsinfected with a wild type vaccinia virus (‘E1/WT’). The lysates werecoated as a 1/20 dilution on a normal ELISA microtiter plate (Nuncmaxisorb) and left to react with a 1/20 diluation of the respectivesera. The panel consisted of 14 type 1a, 38 type 1b, 21 type 2, 21 type3a, and 9 type 4 sera. Human antibodies were subsequently detected by agoat anti-human IgG conjugated with peroxidase and the enzyme activitywas detected. The optical density values of the E1and wild type lysateswere divided and a factor 2 was taken as the cut-off. The results aregiven in the table A. Eleven out of 14 type 1a sera (79%), 25 out of 38type 1b sera (66%), 6 out of 21 (29%), 5 out of 21 (24%), and none ofthe 9 type 4 or the type 5 serum reacted (0%). These experiments clearlyshow the high prevalence of anti-E1antibodies reactive with the type 1E1 protein in patients infected with type 1 (36/52 (69%)) (either type1a or type 1b), but the low prevalence or absence in non-type 1 sera(11/52 (21%)).

TABLE A serum E1/WT type 1a  3748 3.15  3807 3.51  5282 1.99  9321 3.12 9324 2.76  9325 6.12  9326 10.56  9356 1.79  9388 3.5  8366 10.72  83802.27 10925 4.02 10936 5.04 10938 1.36 type 1b  5205 2.25  5222 1.33 5246 1.24  5250 13.58  5493 0.87  5573 1.75  8243 1.77  8244 2.05  83161.21  8358 5.04  9337 14.47  9410 5  9413 5.51 10905 1.26 10919 5.0010928 8.72 10929 8.26 10931 2.3 10932 4.41   44 2.37   45 3.14   46 4.37  47 5.68   48 2.97   49 1.18   50 9.85   51 4.51   52 1.11   53 5.20  54 0.98   55 1.48   56 1.06   57 3.85   58 7.6   59 3.28   60 3.23  61 7.82   62 1.92 type 2   23 0.91   24 1.16   25 2.51   26 0.96   271.20   28 0.96   29 2.58   30 8.05   31 0.92   32 0.82   33 5.75   340.79   35 0.86   36 0.85   37 0.76   38 0.92   39 1.08   40 2.33   412.83   42 1.21   43 0.91 type 3   1 6.88   2 1.47   3 3.06   4 6.52   510.24   6 2.72   7 1.11   8 1.54   9 1.60   10 1.21   11 1.07   12 1.00  13 0.85   14 0.96   15 0.51   16 1.00   17 1.09   18 0.99   19 1.04  20 1.04   21 0.96 type 4   22 0.87 GB48 0.49 GB113 0.68 GB116 0.73GB215 0.52 GB358 0.56 GB359 0.71 GB438 1.08 GB516 1.04 type 5 BE95 0.86

Core/E1 clones of isolates BR36 (type 3a) and BE95 (type 5a) weresubsequently recombined into the viruses wHCV-62 and vvHCV-63,respectively. A genotyped panel of sera was subsequently tested ontocell lysates obtained from RK13 cells infected with the recombinantviruses vvHCV-62 and wHCV-63. Tests were carried out as described aboveand the results are given in the table given below (TABLE B). From theseresults, it can clearly be seen that, although some cross-reactivityoccurs (especially between type 1 and 3), the obtained values of a givenserum are usually higher on its homologous E1 protein than on an E1protein of another genotype. For type 5 sera, none of the 5 sera werereactive on type 1 or 3 E1 proteins, while 3 out of 5 were shown tocontain anti-E1antibodies when tested on their homologous type 5protein. Therefore, in this simple test system, a considerable number ofsera can already be serotyped. Combined with the reactivity totype-specific NS4 epitopes or epitopes derived from other type-specificparts of the HCV polyprotein, a serotyping assay may be developed fordiscriminating the major types of HCV. To overcome the problem ofcross-reactivity, the position of cross-reactive epitopes may bedetermined by someone skilled in the art (e.g. by means of competitionof the reactivity with synthetic peptides), and the epitopes evokingcross-reactivity may be left out of the composition to be included inthe serotyping assay or may be included in sample diluent to outcompetecross-reactive antibodies.

TABLE B serum E1^(1b)/WT E1^(3a)/WT E1^(5a)/WT type 1b 8316 0.89 0.590.80 8358 2.22 2.65 1.96 9337 1.59 0.96 0.93 9410 16.32 9.60 3.62 94139.89 2.91 2.85 10905 1.04 0.96 1.05 10919 3.17 2.56 2.96 10928 4.39 2.282.07 10929 2.95 2.07 2.08 10931 3.11 1.49 2.11 5 0.86 0.86 0.96 6 3.481.32 1.32 7 6.76 4.00 3.77 8 10.88 3.44 4.04 9 1.76 1.88 1.58 10 9.887.48 7.20 11 8.48 8.99 8.45 12 0.76 0.72 0.76 13 5.04 5.67 5.37 14 10.4810.54 11.22 15 5.18 1.62 1.65 type 3 8332 3.39 4.22 0.66 10907 3.24 4.390.96 10908 0.99 0.94 0.98 10934 0.86 0.90 0.90 10927 2.58 2.71 2.44 82100.82 0.80 0.86 8344 1.09 6.66 1.17 8351 1.21 1.29 1.22 30 0.85 4.11 0.9832 0.85 2.16 1.04 type 5 0.78 0.95 1.54 BE110 0.79 1.01 4.95 BE95 0.470.52 0.65 BE111 0.71 0.75 8.33 BE112 1.01 1.27 2.37 BE113 1.11 1.35 1.60

TABLE 5 Homologies of new HCV sequences with other known HCV typesRegion Isolate 1a 1b 2a 2b 3a 3b (nucleotides) (type) HCV-1 HCV-J HC-J6HC-J8 T1 T7 T9 T10 Core (1–573) PC (5) 83.8 (91.6) 84.8 (92.1) 82.6(90.1) 82.4 (89.0) E1 (574–957) HD10 (3) 61.5 (68.0) 64.6 (68.8) 57.8(55.5) 56.3 (59.4) BR36 (3) 62.0 (66.4) 62.5 (67.2) 56.5 (53.9) 55.2(58.6) BR33 (3) 60.7 (67.2) 63.3 (68.0) 56.5 (54.7) 56.0 (58.6) PC (5)61.4 (64.0) 62.4 (64.8) 54.1 (49.6) 53.3 (47.2) GB358 (4a) 62.5 (69.1)62.8 (65.9) 59.4 (54.0) 54.4 (54.0) GB549 (4b) 66.0 (72.2) 62.8 (69.8)59.1 (56.4) 56.5 (54.0) GB809 (4c) 63.3 (69.1) 60.7 (64.3) 56.7 (53.2)53.0 (51.6) NS3 PC (5) 74.7 (89)   76.1 (86.4) 76.1 (89.8) 78.0 (89.0)(3856–4209) NS4 BR36 (3) 67.8 (78.5) 69.8 (75.1) 62.0 (67.5) 61.7 (66.0)(4892–5292) HD 10 (3) 69.8 (74.6) 66.6 (69.7) 57.8 (59.9) 59.1 (59.9)NS4 PC (5) 61.3 (62.2) 63.0 (65.5) 52.9 (46.2) 54.3 (43.7) (4936–5292)NS5b BR34 (3) 65.7     66.7     63.9     64.3     94.8 93.9 75.6 77.0(8023–8235) BR36 (3) 64.3     67.6     64.8     66.7     94.8 93.4 75.176.5 BR33 (3) 65.7     67.1     64.3     64.8     94.8 93.9 76.0 77.5GB358 (4a) 67.7 (76.1) 65.6 (77.0) 66.5 (70.8) 65.6 (71.7) GB549 (4b)68.8 (76.1) 67.1 (77.0) 65.9 (71.7) 65.9 (74.4) GB809 (4c) 68.5 (73.5)65.0 (73.5) 67.7 (69.9) 67.7 (73.5) Shown are the nucleotide homologies(the amino-acid homology is given between brackets) for the regionindicated in the left column.

TABLE 6 NS4 sequences of the different genotypes SYNTHETIC PEPTIDESYNTHETIC PEPTIDE SYNTHETIC PEPTIDE prototype TYPE NS4-1 (NS4a) NS4-5(NS4b) NS4-7 (NS4b) position-> 1 1 1 1 1 1 6 7 7 7 7 7 9 0 2 3 3 4 0 0 00 0 0         * *  *    **   **       *       *   *  *  *    *   *  **HCV-1 1a LSG KPAIIPDREV   LYREFDE SQHLPYIEQ GMMLAEQFKQ KLAEQFKQ  KALGLLQTAS RQA (SEQ ID NO:272) (SEQ ID NO:273) (SEQ ID NO:274)HCV-J 1b LSG RPAVIPDREV  LYQEFDE ASHLPYIEQ GMQLAEQFKQ KLAEQFKQ  KALGLLQTAT KQA (SEQ ID NO:275) (SEQ ID NO:276) (SEQ ID NO:277)HC-J6 2a VNQ RAVVAPDKEV LYEAFDE ASRAALIEE  GQRIAEMLKS KIAEMLKS  KIQGLLQQAS KQA (SEQ ID NO:278) (SEQ ID NO:279) (SEQ ID NO:280)HC-J8 2b LND RVVVAPDKEI  LYEAFDE ASKAALIEE GQRMAEMLKS KMAEMLKS KIQGLLQQAT  RQA (SEQ ID NO:281) (SEQ ID NO:282) (SEQ ID NO:283)BR36 3a LGG KPAIVPDKEV LYQ

YDE SQAAPYIEQ  AQVIAHQFK

 K IAHQFK

 K

LGLLQRAT 

QQ (SEQ ID NO:97) (SEQ ID NO:99) (SEQ ID NO:100) PC 5LSG KFAIIPDREA  LYQ

FDE AASLPYMDE  TRAIAGQFK

 K IAGQFK

 K

LGFISTTG  

KA              V (SEQ ID NO:284) (SEQ ID NO:105) (SEQ ID NO:102) and(SEQ ID NO:103), respectively *residues conserved in every genotype.Double underlined amino acids are type-specific, amino acids in italicsare unique to type 3 and 5 sequences.

TABLE 7 SEQ ID Primer NO NO (polarity) Sequence from 5′ to 3′ 63HCPr161(+) 5′-ACCGGAGGCCAGGAGAGTGATCTCCTCC-3′ 64 HCPr162(−)5′-GGGCTGCTCTATCCTCATCGACGCCATC-3′ 65 HCPr163(+)5′-GCCAGAGGCTCGGAAGGCGATCAGCGCT-3′ 66 HCPr164(−)5′-GAGCTGCTCTGTCCTCCTCGACGCCGCA-3′ 67 HCPr23(+)5′-CTCATGGGGTACATTCCGCT-3′ 68 HCPr54(−)5′-CTATTACCAGTTCATCATCATATCCCA-3′ 69 HCPr116(+)5′-ttttAAATACATCATGRCITGYATG-3′ 70 HCPr66(−)5′-ctattaTTGTATCCCRCTGATGAARTTCCACAT-3′ 71 HCPr118(−)5′actagtcgactaYTGIATICCRCTIATRWARTTCCACAT-3′ 72 HCPr117(+)5′-ttttAAATACATCGCIRCITGCATGCA-3′ 73 HCPr119(−)5′-actagtcgactaRTTIGCIATIAGCCKRTTCATCCAYTG-3′ 74 HCPr131(+)5′-ggaattctagaCCITCITGGGAYGARAYITGGAARTG-3′ 75 HCPr130(+)5′-ggaattctagACIGCITAYCARGCIACIGTITGYGC-3′ 76 HCPr134(+)5′-CATATAGATGCCCACTTCCTATC-3′ 77 HCPr3(+) 5′-GTGTGCCAGGACCATC-3′ 78HCPr4(−) 5′-GACATGCATGTCATGATGTA-3′ 79 HCPr152(+)5′-TACGCCTCTTCTATATCGGTTGGGGCCTG-3′ 80 HCPr52(+)5′-atgTTGGGTAAGGTCATCGATACCCT-3′ 81 HCPr41(+)5′-CCCGGGAGGTCTCGTAGACCGTGCA-3′ 82 HCPr40(−)5′-ctattaAAGATAGAGAAAGAGCAACCGGG-3′ 124  HCPR2065′-tggggatcccgtatgatacccgctgctttga-3′ 125  HCPR2075′-ggcggaattcctggtcatagcctccgtgaa-3′ 141  HCPR1095′-tgggatatgatgatgaactggtc-3′ 142  HCPR14 5′-ccaggtacaaccgaaccaattgcc-3′

TABLE 8 NS4 SEROTYPING Type 1 NS4 Type 2 NS4 Type 3 NS4 serum 1 5 7 1 57 1 5 7 type 1a 101 3 3 3 − 1 3 +/− +/− 3 102 1 +/− 2 − − 2 − − 1 103 13 3 − +/− 3 − +/− 3 104 3 3 3 2 2 3 3 +/− 2 105 3 3 3 − 2 2 +/− +/− 2106 3 1 1 − 1 2 +/− +/− +/− 107 3 3 3 − 2 2 2 − 1 108 3 3 3 − +/− 2 +/−1 2 109 3 3 3 +/− 2 3 1 − 3 110 3 3 3 − +/− 1 − − 3 type 1b 111 +/− +/−− − − − − − − 112 − 2 3 − − 2 − − 3 113 2 3 3 − − 1 − − 3 114 2 3 3 1 +2 + 1 3 115 3 3 3 − + 3 − − 3 116 3 3 3 − +/− 1 − − 1 117 3 − − 3 +/−+/− +/− − − 118 1 2 3 − +/− 2 − +/− 3 119 +/− 2 2 +/− +/− 2 + 1 2 120 −3 3 −3 +/− +/− − − − 121 3 3 3 +/− 2 2 2 2 3 122 3 3 1 − 1 2 2 1 1 123 33 2 − 1 2 − 1 1 124 3 3 3 +/− 2 − − 2 125 3 3 3 1 1 3 2 1 3 126 1 2 2 11 1 1 1 1 127 3 2 +/− − +/− 1 +/− +/− +/− 128 3 3 3 − +/− 1 2 +/− +/−129 2 3 3 − − 3 − − 3 130 − 2 1 +/− − − − − − 131 − 1 1 − − − − − +/−132 − − − +/− − +/− +/− − − 133 3 3 3 − 1 3 − 1 3 134 − 2 2 − − − − − −135 3 3 3 1 + 2 2 1 3 136 − 3 3 +/− +/− +/− +/− − 3 137 +/− +/− +/− +/−+/− +/− +/− − − 138 3 3 3 +/− 2 2 1 + 3 type 2a 139 3 − − 3 3 +/− 1 − −140 +/− − − 3 3 3 3 − − 141 2 − − 2 1 +/− 2 − − 142 − − − − +/− − − − −143 − +/− +/− 1 2 1 1 +/− +/− 144 1 1 + 1 3 2 1 1 2 145 − +/− +/− 3 1 22 +/− +/− 146 − − − +/− +/− − − − − 147 − +/− − 3 1 3 − − − 148 − − −+/− − − +/− − − type 2b 149 − +/− +/− 3 3 1 2 +/− +/− type 3 150 +/− +/−+/− +/− +/− +/− 1 3 3 151 − − − − − − 2 − 2 152 +/− − − − − − 3 − − 153− − − − − − − 1 − 154 +/− 1 3 − +/− 2 2 1 3 155 − 2 3 − 2 2 1 1 3 156 −− − − − − − − − 157 − − − +/− +/− − +/− 2 2 158 2 − − − 1 2 3 2 2 159 −− − − +/− +/− − 3 3 160 − − − − +/− − − 2 3 161 − − − − 1 1 +/− 3 2 type4 162 1 − − − − − − − − 163 2 − − − +/− +/− +/− − −

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1. An isolated HCV antibody which specifically binds to a type 3a HCVantigen consisting of 5 or more contiguous amino acids selected from theregion spanning positions 140 to 191 of the Core region of HCV type 3a,wherein the antigen contains at least one HCV genotype 3 a-specificamino acid.
 2. The HCV antibody according to claim 1 wherein said regionspanning positions 140 to 191 of the Core region of HCV type 3a is anyone of SEQ ID NOs: 14, 16, 18, 20, and
 24. 3. The HCV antibody accordingto claim 1 which has been produced upon immunization of a mammal withsaid antigen.
 4. The HCV antibody according to claim 1 which is amonoclonal antibody.
 5. A humanized version of an HCV antibody accordingto claim
 4. 6. The humanized version of an HCV antibody according toclaim 5 which has been humanized by means of recombinant DNA technology.7. The HCV antibody according to claim 1 which further comprising alabel.
 8. The HCV antibody according to claim 7 wherein said label is ofthe enzymatic, fluorescent or radioactive type.
 9. A compositioncomprising an HCV antibody according to claim
 1. 10. A kit fordetermining the presence of HCV antigens present in a biological sample,said kit comprising: (a) at least one HCV antibody according to claim 1,(b) a buffer enabling the binding reaction between an HCV antibody of(a) and an HCV antigen present in said biological sample; or componentsnecessary for producing said buffer, (c) a means for detecting theimmune complexes formed between an HCV antibody of (a) and an HCVantigen present in said biological sample.
 11. A kit for determining thepresence of HCV antigens present in a biological sample, said kitcomprising at least one HCV antibody according to claim
 1. 12. A methodfor determining the presence of HCV antigens present in a biologicalsample, said method comprising the steps of: (a) contacting saidbiological sample with at least one HCV antibody according to claim 1(b) detecting the immune complexes formed in (a), (c) inferring from (b)the presence of said HCV antigens in said biological sample.